Science topic

Histology - Science topic

The study of the microscopic anatomy of cells and tissues of plants and animals, including tissue fixing, fixation and staining.
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Although the donut concept of the optic disc is widely published in glaucoma but it is not supported by any histology in the textbooks.
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Erin Denny I believe that if the neuro-retinal rim concept was correct, then I should have found an optic disc histology depicting the ‘donut’ appearance. I still have not come across any such image in the textbooks during my 60 years in ophthalmology. Thank you for the helpful information. I appreciate your kind response.
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Nanoparticles impact on animal tissues:
Histological (H&E) analysis of liver tissues, revealed the presence of slightly orange-colored cells.......
Does anybody observe similar structures in liver tissues?
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Please, send me a photo (photos).
Best,
Peter
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I'm trying to identify a marker/dye that can be assessed (via histology) from post-mortem eyes which have undergone subretinal and suprachoroidal injections. Any advice on successful agents would be useful.
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Dear Andrew,
Years ago I used to use 5% Pontamine Sky Blue (in 0.5 M sodium acetate) dye in vivo in rat brains to label the location of extracellular unit recording sites. You will need to apply 5-10 microAmp currents for about 10 minutes. You can visualize permenant dye deposits as blue dots in at the injection. It is relatively cheap and easy to visualize with a light microscope. For details of the methods please see Methods section in the following publication.
best wishes,
Refik
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do T or B lymphocyte s present in histological structure of ovary ?what is the defence mechanism in ovary ?
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pH. of ovaries provide protection against various microorganism. Beside that ,hormones provide nourishment to ovarian cells. Egg is released from ovaries about three months
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What type of study (or studies) could prove or disprove the hypothesis of parthenocarpy in the Cannabis L. plant genus?
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Andrew Paul McKenzie Pegman I published such observations in my paper Cannabis Ontologies 1 from 2020, but it has not been taken upon and cannabis continues to be described as a flower— so there is a need for some more proof to be advanced
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This is a histologic picture of the spermatheca of a male fish, what is this part of the picture in red?
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Dear Sir,
The attached paper might be helpful for you.
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Hey everyone, has anyone ever performed immunohistochemical staining followed by histochemical staining on the same histological section (like a double labeling)?
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I will do on two adjacent sections, on for histology and other for IHC. that will be better
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Dear Everyone!
I am looking for an illustrated paper, showing a cross-section view of a Myliobatiformes tooth with indicating the characteristic tissue-types. I there any paper out there in this topic?
Thank you; Márton
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Hi Marton,
I just saw your question of June 28...
In addition to the replies by Patrick & Jürg:
Check Agassiz's Poissons fossiles, plate volume 3, his "Tab. R", figs 1-2.
And another interesting paper on this topic might be Harless, 1849 - Ueber den Zahnbau von Myliobatis.
I have a spare original hard copy of this paper. If interested, just let me know. I can send it to you.
Cheers,
Frederik
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Frozen sectioning via microtome, sections 40 um thick.
I am wondering if it looks like the sections are thinner in those ventral, whiter spots (perhaps from the tissue warming too much in that spot?)--or, perhaps, a dehydration artifact, if perhaps these sections were sticking out a bit from the cryo solution in the tube or during washes. These two sections are 10 sections (400 microns) apart from each other. Or a freezing artifact -- but other sections look fine.
I notice the very edges of sections similarly turn opaque white sometimes as they dry. Maybe these points are slightly lifted off the slide?
Thank you for your help!
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Thank you so much, it could be some debris and air bubbles, as well as an adherent of gelatine particles
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Spermatogonia cells , Necrosis
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I would like to request you to go through the published Paper- McClusky LM. Several routes of cell death to secondary necrosis in the elasmobranch testes. Apoptosis 2022; 27:454-464.
The paper will be helpful to get your question answered.
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Hi, I am currently working with pancreas. I had stored pancreas at -80. Can I thaw the tissue and use it for histology purposes ? Or is there any alternative ways to check the level of beta pancreatic cell ?
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The published paper of Joosten L, et al. Mol Pharmaceutics 2019;16,9:4020-4030; may be gone through for this purpose. The authors had done the experiment in NOD mice.
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Hello there. I just completed an experiment using both hM4D(Gi) and hM4D(Gq) DREADDs in the cerebellar vermis, both co-expressed with mCherry. I noticed that while I had a lot of expression of the Gi DREADD in my histology, there was little to no expression of the Gq. Has anyone else observed this/ seen articles where this may be the case? Thank you!
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Activation of hM4D reduces membrane excitability. Several Studies had been made regarding its expression in hippocampus which is the temporal extension of cerebrum. The selective expression of hMRD(Gi)_in principal hippocampal neurons have seizure suppressing effects. Categorical quantitative expression of it in cerebellum had not been studied. So comparative conclusion may not be possible. Might be, its expression in hippocampus is very important.
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Dear colleagues - i am working with temporal bone histology prepared in celloidin. The slices are about 20 microns thick and were stained with H&E. The material was digitized and saved both in JPG and Raw formats. I am looking for a program that can do 3D reconstructions, preferably straight from the histology images (meaning no need to pre-delineate the areas of interest). Does anyone have any program recommendation or experience with this type of work? Appreciate your comments! Thanks! Miriam
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3D histology reconstruction as you know has the great potential to have a view of structures in the spatial arrangement having more convenient interpretation of tissues. In fact, it has its root in embryology. I would like to request to search online to obtain detailed information in this regard.
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I am currently repeating the same procedures with slightly different methods, but every time, my brain tissue slices disappear from the slides within a few weeks of coverslipping them. The slides have perfect brain tissue outlines where there are greyish debris-like material (maybe?) at everywhere outside of the slices and where the ventricles were, but where the tissue was is crystal clear.
Here are the steps I take:
1. Perfusion with 4% PFA and transferal of brains into the same PFA solution
2. Transferal of brains to 30% sucrose for 3 days
3. Section tissue via microtome and store in 96 well plates with PB+Azide
4. Transfer select tissue to gelatinous mounting solution to help place tissue onto the top of polarized slides
5. Let tissue on slides dry for two days
6. Wash the slides
  • For some slides, the process included the consecutive rinsing with higher percentages of alcohol and then with xylenes
  • For others, the process only involved rinsing with deionized water for 2 minutes
7. Add 50 uL of the Vectashield DAPI mounting media onto the slides and add coverslips at an angle to prevent bubbles
  • For some slides, hardening version of the mounting media was used
  • For others, the non-hardening version of it was used and nail polish was added to the edge of coverslips to prevent sliding
Please help. I only have a few days to figure this out and none of my lab members have seen anything like this before and I would like to prevent this from happening.
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1) If you're using PFA, don't do a sucrose saturation.
2) If you're going to do a sucrose saturation, start with a 5% sucrose solution then a 30%.
3) Finally, as Ute and Nabanita have suggested, use SuperFrost + slides, skip the vectashield, and keep slides in the fridge for storage.
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any scoring system available or a quantitively method?
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Thanks for replying, do you have any refrence for that scoring?
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What would be the best immunostaining modality when looking to prepare histology slides from the female periurethral tissue? The goal is to determine whether neurovascular and glandular tissues can be isolated from the anterior vaginal wall. Is it better to use immunohistochemistry or immunofluorescence, or perhaps another method?
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Detection of a specific antigen in tissues is known as immunohistochemistry. It has following procedure. 1-To deparaffinize the tissue, 2- To rehydrate it ,3- To apply primary antibody. 4-To apply enzyme-conjugated secondary antibodies, 5- Specific staining after adding enzyme-specific substrate. Detailed information might be obtained from searching internet.
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Dear All,
I am wondering how the Transwell inserts can be recovered from fixing to be then embedded?
After fixing, I put each membrane on nitrocellulose membrane to avoid curling and then I leave them in an embedding cassette to be processed - automated protocol of our histology facility.
When embedding, I cut each membrane into half and put them upright. This process is very difficult because the Transwells start curling and they are very thin, so they don't settle very well. Also, when sectioning, Transwells would break because of their thickness.
Do you have any alternative on how to manage them before processing and then embed?
Thank you!
Aurora
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Transwell inserts are permeable support device meant for the study of cell-lines to study angiogenic inducing or inhibiting effect on the migratory response of epithelial cells. I would request you to go through the publication of Sip CG, et al. Lap Chip 2014; 14:302-314; where it had been described with method compatible with conventional cell culture.
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Hello all!!
I am currently quantifying damage in renal histological sections of AKI model. I found EGTI scoring system during the literature search. However, I did not find the detailed protocol of it. Does anybody help me with the step-by-step protocol of this scoring system?
I really appreciate any help you can provide.
Sreyasi!
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EGTI is a reliable and informative assay to determine the degree of damage arising out of endothelial, glomerular, tubular and interstitial injury and hence utilized for reporting histological damage caused by kidney ischemia. However, the detailed method of calculation might be obtained by searching internet.
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Hi there,
I am looking for the number of fibroblast in healthy human skin- dermal fibroblasts and I am not dividing them into subpopulations.
Does anyone have a good reference for this?
Optimal would be a value in [cells/cm^2 skin]. Maybe from histological countings or from isolations- How many fibroblasts can be isolated per cm^2?
It would be awesome if someone had an answer :-)
Kind regards
Miriam
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Fibroblasts are primarily present in dermis of skin. The dermis again can be divided into superficial papillary layer and lower reticular layer. The papillary layer is having high density of fibroblast in a soft connective tissue background, while the reticular layer contains lesser fibroblast in a fibrous connective tissue background. However, the exact number and concentration of fibroblasts in the skin have not been mentioned categorically in the literature.
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I want to study the male reproductive system for two different seasons to obtain histology, histometery, morphology and morphometery. Please guide me how much should be my simple size. What would be a statistically robust sample size?
I plan to take 6-11 animals per season based on my calculation using the Animal research equation using resource equation approach- group comparisonbut some statisians say to use g power analysis.
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If you plan to study only the testes, it is not the male reproductive system. In any case, how you will cut the specimens to be processed, and which embedding orientation of the specimen and cutting procedure will be implemented? You are looking already at the end phase but there is a lot in between...Imagine if I would be a reviewer of your manuscript and you did not write anything in the M&M sections...A lot of more details to be provided.
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The slides are not deparaffinized yet. I'm using a scalpel to scrape off the tumor area from it, however, the area of interest is very small. I had a hard time transferring the scrapping piece into the tube. I saw some youtube videos, they pre-fill the Eppendorf tube with 100% ethanol, then immerse the scalpel into the liquid to ensure all the pieces are collected. Is this a proper way to do it? , if so, how would I get rid of ethanol and proceed with the deparaffinized process?
My next step is to deparaffinized FFPE and extract RNA and DNA with Qiagen kit. Do you think I can pre-fill deparaffinized solution into the tube, and then immerse my scalpel into the solution to collect all the tissue pieces?
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Pure ethanol will evaporate if you leave the tube open. It does take a while though.
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My lab has recently be discovering a problem that some of our old nissl stain (2-3 years old) is turning to a disgusting orange/ brown color. Its not even every slide that was stained together, just a few every once in a while. Our slides are coverslipped with permount and glass and there's no air bubbles in any of the slides
Does anyone have any idea what could be causing this?
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Hello Jessica,
can you give some more details about the staining protocol and how you have prepared the staining solution?
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I have unstained FFPE tumor tissue, with the area of interest graphing on the back of the slides. The goal is to scrap off the area of interest, then extract both DNA and RNA.
What's the good order of the procedures?
Should I scrape the tissue first and then deparaffinized it? Someone also suggests me to bake the slides first and then scrape them.
Appreciate any pov!
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Hi Yun,
Generally, when using the whole slide, scraping is done after rehydration (post-xylene etc.). However, based on your question it looks like you do not want the whole section.
Right at the beginning before deparaffinization, you could use a very sharp blade and then cut around the area you want. Don't scrape it or anything and leave the area still attached. Make sure you make a good cut through the tissue. This would the best time to make the cut when the slide is untouched and section is firm. Then you can do deparaffinization with xylene etc. and when you finish, the rehydration step, it will be easier to just remove that area by scraping, or use a regular sharp shaving blade to "lift" off the area.
Trying to cut after the rehydration step you will definitely be dealing with more fragile tissue and cutting may not be that easy. Don't do too much baking etc. this will jsut degrade the RNA/DNA that much more.
Hope this helps!
Good luck!
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Hi everybody,
I am currently conducting some pilot experiments where I perform intratracheal instillations of LPS in rats to induce pulmonary inflammation. My plan is to examine some morphological characteristics in the left lung of the rats to verify the LPS-induced inflammation (via H&E staining and/or identification of protein membrane of macrophages/infiltrating neutrophils). However, I am a bit hesitant with regards to the optimal cryopreservation technique (although FFPE sectioning is commonly done for that kind of analysis, I prefer for several reasons to examine first whether I can do frozen tissue sectioning).
As far as I understand, one of the good ways to cryopreserve lung architecture is to inflate lungs with OCT (either pure or diluted 1:1 with PBS) to prevent atelectasis, then embed the tissue entirely in OCT and finally freeze it at supercooled isopentane or preferably n-heptane for rapid freezing before storage at -80 C.
So when the OCT intratracheal instillation should be carried out? Should you do it in situ at necropsy or do you have to remove the whole complex (trachea + lower airways) and infuse the trachea? I am sceptical whether the latter (removal of the whole complex) could lead to some extent of irreversible deflation even after the subsequent inflation with OCT.
Also, for how long do you embed the inflated lungs in OCT and in what temperature? I understand that this step is for interfering with the van der Waals forces to mitigate ice crystal formation but I was thinking whether after embedding you should remove extra OCT. Could any extra OCT ,surrounding the tissue, lead to freezing artefacts due to insulation and subsequently slower freezing?
Finally, do you usually supercool n-heptane in liquid nitrogen? and how do you understand it has reached optimal freezing temperature?
I would appreciate a lot any kind of advice,
Thanks a lot in advance
Angelo
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I've not done much examination of frozen lung sections. The large majority of our work is done on FFPE tissue, we only work on frozen tissue in the rare cases where we're looking at IHC/IF for markers that don't work on FFPE. If you're looking for good morphology preservation, you might be disappointed with frozen sections, which are much more difficult to do and require a fair bit of training to do well, and even then are often prone to artifacts.
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Liver histology
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I have paper about this in my page
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Liver histology
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Please find the attached links, you can check the histopathological and anatomical differences between human and rat liver.
Thanks
Samir Ranjan
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When staining with hematoxylin and eosin of a muscle biopsy from a patient with T341P desminopathy, we observe accumulations of inclusions similar to nuclei (arrows in figures 1 and 2, x280). And outside of these accumulations - adipose tissue, which used to be muscle tissue. There are no such massive accumulations of inclusions in adjacent muscle fibers. We assume that clusters of inclusions are not nuclei? Figure 2 is the inverted figure 1.
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Dear Geir Bjorklund, Duc M. Hoang, John Hildyard, thank you very much for your answers and recommendations!
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Has this ever happened to anyone? If so what did you do?
Hello, I have done immunohistology for human and rat tissues, both chromogenic and fluorescent staining. The tissues of FFPE and I go through the normal steps of removing the paraffin, rehydrating the samples, antigen retrieval (HIER), blocking (peroxidase for chromogen, serum for the secondary), primary antibody addition and so on. I rarely get good signals. I am now working with a new protocol from a company dealing in fluorescent probes and I followed their protocol to the letter, and still no concrete signal. The fluorophore is red 594 but I get almost no signal in the red channel and a some signal in the green channel. When I overlap the green and red are always in the same place.
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There could be a number of reasons for weak or absent signals in immunohistochemistry, including:
  1. Antibody quality: Make sure the primary antibody you are using is validated for IHC, fresh, and appropriate for the species and tissue type you are working with.
  2. Antibody concentration: The antibody concentration may need to be adjusted to optimize staining. Sometime you may need as high as 1:15 dilution for primary and 1:100 for secondary.
  3. Antigen retrieval: The antigen retrieval method and time may need to be adjusted, or a different method may be more effective.
  4. Blocking: The blocking step is critical for reducing background noise. Ensure that the blocking reagents and conditions are appropriate.
  5. Detection system: The detection system, including the secondary antibody and detection reagents, may need to be adjusted or replaced to ensure compatibility with the primary antibody and tissue type.
  6. Staining protocol: Ensure that the staining protocol, including incubation times and temperatures, are followed exactly as recommended.
  7. Tissue preparation: The quality of the tissue samples and how they were prepared can affect staining results. Make sure the tissue samples are fresh and well-fixed, and that the tissue is thin enough to allow for proper staining penetration.
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Hello! im about to start an immunohistochemistry assay to analize fetal brains (day 15) with different treatments. I have the head samples (because i cant obtain the intact brain with LPS treatment) and im about to start with the dehydration protocol, but im not sure how much time should i leave the samples in every step. Does anybody use this kind of sample?
Ty a lot!
Protocol for dehydratation of Mouse fetal head for Paraffin embedding
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thank you for sharing Dr. Juli, I think you can use the same protocol for dehydration of other tissues
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Hello research enthusiasts!
I am looking for histological changes in the rat liver (inflammatory changes, fat vesicles etc) induced by a specific compound. I would be very thankful if anyone can suggest me some books with good detalls on the histology as well as pathology of rats (my target is liver, brain and GI sections)
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The problem is not the liver structure which is similar to humans, but how the rat liver will respond to your compound compared to humans and the experience that you have as clinical/experimental pathologist (it looks from your message that you are starting from scratch). It is not possible that you can have the same level of experience in liver, GI, and brain. Are three areas of different expertise which requires years of training in the clinical area. For the brain in particular, there is no atlas with cell identification and distribution available, in stark contrast with the clinical picture.
Especially if the study aims at translational applications, I would suggest that you confirm your results with a pathologist for each subspecialty in the academic center and ask also for suggestions. I have personal experience in many experimental papers in which the images do not match the findings described in plots, even if published in prestigious journals.
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I am studying Salmonella infection in mice. At the endpoint our study we harvest the liver, spleen, kidneys and small intestines' from infected mice. We want to look at Salmonella dissemination, inflammation and immune cell infiltration. I have come across many papers that look at the liver and spleen via histology.
I was wondering of the 4 organs I've harvested which one may be the best for histological analysis.
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However, based on the information you provided, the spleen and liver are likely to be the best organs to look at for histological analysis of Salmonella infection and dissemination in mice. Both the spleen and liver are primary sites of bacterial replication and accumulation, and will likely show the most pronounced signs of infection and inflammation. Additionally, these organs can provide information on the host immune response to the infection.
The small intestine and kidneys may also be informative to examine, as the small intestine is the main site of entry for Salmonella and the kidneys may also be affected by the infection.
It is important to note that the most informative organ may depend on the specific strain of Salmonella and the mouse model being used. It's recommendable to analyze all the organs that you have harvested to get a comprehensive view of the infection process, and to compare the different findings to understand the dissemination and host response.
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Hi everybody,
I am experiencing some troubles in my fish gonad samples included in paraffin, and I would like to ask you some advices.
The protocol I am using has been already used for years (sea urchin gonads, mullet gonads, oysters...) and it has always worked well. The only things I changed are the type of paraffin (now I am using Leica Paraplast 56°C, before a paraffin with higher melting point) and I started to make practice with the histomolds (plastic cassettes + metal molds).
Recently, I included some small pieces of mullet gonads (4x3x2 mm, more or less) and I tried to cut them at 5 microns. I succeded in creating the ribbon, but when the blade met the tissue, it crumbled. Tissue seems very hard in some samples (like crystallized) but it seems normal in others, and the slices came out with a hole in correspondence with the tissue.
I don't know if I failed the infiltration (oven set at 59°C), or if the dehydration - clearing steps are wrong. The strange thing is that the protocol has always worked perfectly, with small or bigger tissues.
In your opinion, what am I doing wrong? I attach the whole protocol below.
fixation in 10% formalin
wash in water
70% alcohol (1 change/day, x2) before processing
Processing:
1h 70% alcohol
1h 80% alcohol
1h 96% alcohol
1h 100% alcohol
1h 100% alcohol
1h 1:1 100% alcohol : Bioclear (xylene substitute, natural terpenes)
30 min Bioclear
30 min Bioclear (in oven at 59°C)
Paraffin (Paraplast) overnight (in oven at 59°C)
3h new Paraffin (in oven at 59°C)
inclusion RT
You may consider that we do not have an automatic tissue processor so we have the need to block the protocol in the evening. Even for the inclusion step, we don't have neither paraffin dispenser nor embedding station. I store my melted paraffin in the oven, I pour it in the metal mold positioned on a hot plate (paraffin remains very hot and melted), I put the piece of tissue in the mold, I transfer the mold on a reusable ice pack to create a thin solidification of paraffin, then I put a plastic cassette on top, I fill it with more melted paraffin and I put all on the ice pack to cool fast.
Any suggestion?
Thanks in advance.
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I just wanted to comment that I was having the same issue with liver samples crumbling (like sandstone) during sectioning and soaking the tissue blocks in water for the 30min - 1hr made a huge difference. The water seems to hydrate the tissue allowing it to stay in tact during sectioning. If it starts doing it again I plop back into the graduated cylinder with dH20 for a few mins. The tissue sample will protrude from the block some, so you may not want to leave it in water for too long because with the first pass of the blade you will lose some of your tissue. Thanks Omar! The animal tissue techniques that you cited is great. I will be referring back to it whenever I run into anymore issues!
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Hello,
I would like to know whether the following compounds can be used interchangeably for Van Gieson staining:
Many thanks!
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Admitting to have done some staining applications with acid fuchsin(e)
(CI: 42685), formerly (in my active work-life):
Dear James Oakes , at least a short search in some sources available either in Histo-books, or in www (especially SDB's /TDS's / >specifications< from main suppliers (like SIGMA-Chemicals or others) will reveal that Acid fuchsine Ca-salt is a 'protocolled' candidate (also 'certified by the Biological Stain Commission', cf.: https://biologicalstaincommission.org/protocols/:
(Copy and Paste:)
ACID FUCHSINE C.I. 42685
Properties
UV-VIS range: 542-546
Min. Dye Content: 60%
Molecular Formula: C20H17N3Na2O9S3 or C20H17CaN3O9S3
Formula Weight: 585.55 or 579.65
Appearance: Green Crystaline Powder
Dye Class: Aminotriarylmethane
Biological Applications: Widely used plasma stain, which also has many other uses including staining connective tissue, mitochondria, and unaborted pollen grains.
Commercial Applications: Textile Dye, dyeing of crepe paper, soap, and photographic film.) (-End of citation-).
Sigma's product page informs:
"certified by the Biological Stain Commission, Dye content ≥60 %
Synonym(e): Fuchsin S Calciumsalz, Rubin S
Lineare Formel: C20H17N3O9S3Ca MG: 579.64
Solubility: CLEAR RED TO VIOLET SOLUTION AT 1 MG PLUS 1ML OF WATER
Certified for use in Van Gieson connective tissue stain, Mallory′s connective tissue stain and Altmann′s mitochondrial stain."
ThermoScientific (Fisher):
Thermo Scientific™ Saures Fuchsin-Natriumsalz
Summenformel: C20H19N3Na2O9S3
Molekulargewicht (g/mol)
587.544
Acid Fuchsin sodium salt is used as a pH indicator. It is one of the dyes used in Masson’s Trichome Stain where it is used to distinguish muscle from collagen. Solubility in water: (1 mg/ml)
Synonyms: acid fuchsin, acidal fuchsine, acid fuchsine, acidal magenta, fuchsine acid, fuchsin acid, p-fuchsine acid, acid fuchsine n, acid fuchsine o, acid fuchsine s
>>Acid fuchsin calcium salt (CAS 123334-10-1), a stain certified for use in Altmann′s mitochondrial stain, from Santa Cruz….Alternative names/synonyms: ==>Acid Magenta; Acid Rubin
Acid fuchsin calcium salt is certified to use in Altmann′s mitochondrial stain, Mallory′s connective tissue stain and Van Gieson connective tissue stain.[MW: 579.63 / MF: C20H17N3O9S3 Ca
Literaturhinweise / References:
1. Penney, D.P., et al. 2002. Biotech. Histochem. 77: 237-275. PMID: 12564600
Hopefully you will/are able top cope with the handling of the needed picric acid (;-))...
[Van Gieson Stain - VGS:
Picric Acid , saturated aqueous solution.....50 ml
1.0% acid fuchsin, aqueous solution ........... 9 ml
A. dest. / distilled water ...............................50 ml
Learning about the properties / description of Acid fuchsin(e), as described in CONN's Biological Stains(A Handbook of Dyes, Stains, and Fluorochromes for Use in Biology and Medicine), 10th Ed.; ed. by R.W HOROBIN and J.A. KIERNAN (2002 Published for the Biological Stain Commission by BIOS, Oxford UK) on further request.
From Salzburg, Austria - My best wishes...and good luck, WHM.
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is mitophagy a subtype of autophagy?
after reading papers I still do not get a clear anxwer. Some reported that with the increase of  overall autopahgy, mitophagy also increases. However, other argued that these two are actually competing each other.
any one can clarify this?
thanks
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I had a question about why you are studying mitophagy specifically over autophagy in general? I have a collaborator that wants to develop a role of mitophagy in a therapy but we already know autophagy is playing a role, so what is the point of mitophagy specifically? Since it is just the specific degradation of mitochondria.
Thank you,
Ryan
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Hello, there are some holes/porous structure on my nissl staining results as shown in the picture below. Does it look like freezing defects or slides over dried? Some advice will be appreciate. The procedure is described below :
The mouse was perfused fixed with pbs and 4% pfa and then head was left in 30% surcose pfa solution (w/v) for very long time( I know this looks weird but previous lab member could get decent Nissl staining results from it). Then the mouse head was frozen in -80 and brain was extracted with ephys probe embedded inside. Afterwards brain was embedded in oct and put in -80 for freezing and preserving. Frozen oct block is frozen sectioned, dried for 12hrs at RT and nissl stained(5min xylene defat, descending alcohol 3min rehydrate, di water 1min, toludine blue 4 min, ascending alcohol for differenti and xylene 30min for clearing.
Thanks
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*Are you observing similar holes/porous structure in all the samples? If yes then is the temperature of the cryostat optimum?
*Were the sections of 40 micron? I suppose it may be due to the slices being thin.
*I would not recommend keeping the entire head in sucrose as there may be proper permeability issues and since sucrose play a key role in protecting the tissue integrity.
*How I would recommend is to Anesthetize the animal and perfuse intracardially with 4% PFA. Following perfusion collect the brain and store it in 4% PFA at 4 degree (48hr) or room temp for a few days. After Fixation, the brains have to be kept in gradient sucrose (10, 20 and 30%) until the brain tissue settles below the container. If you are using a cryotome, prepare blocks using OCT medium by snap freezing in liquid nitrogen, cut 40micron sections of the brain and dry the slides for overnight.
*Staining may not be an issue as the neurons look great.
Hope it helps
Thanks and Regards
Samir
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Other sustainable alternatives for a histology lab are welcome.
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Try using Histochoice products (clearing and adhesive).
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Hi, histotech colleagues. My name is Anna and I'm a vet pathologist from Ukraine. Now I am looking for specific stains for my work. Van Gieson, Wartin Starry, PAS, and others. May you advise me on the label and manufacturer of stain, that you are usually using in work and with good results. I used stain produced by Diapath in the past. But it didn't meet my needs, unfortunately(
What particular label of histochemical stain do you usually use in the lab? Many thanks in advance.
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Hi Anna,
if you have protocols of the staining procedures and recipes how to prepare the dye solutions then it is not so difficult.
If you have recipes and you have to buy the dye substances , you can buy them as Sven recommands , at Sigma-Aldrich / Merck. Helpful is the Colour Index (C.I.) of the dye, especially when different names are used. In combination with this C.I. No. most the time it is mentioned whether the substance is suitable for histologigacal stainings.
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I would like to fix and stain the eye tissue to observe vascularization on the iris. One thing that should be noted is that I want the anterior chamber space to be intact in terms of volume. Is there any fixation method that would be helpful for this?
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If you do not need the retina, you can cut the posterior eye cup along the ora serata away. Put then the anterior eye part in the fixative of your choice. The advantage is that the anterior part of the eye will be fixed from both sides.
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Dear Researchers:
How to interpret the huge similarity between the network of the universe and the histology building of the human brain?
Is there any relationship or association? What do you think?
Please leave your scientific comment here...
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String theory.
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Formalin is commonly used preservative for routine histology.
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Wonder how many new posters can offer the same information already posted numerous times.
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Hello
I am using KEYENCE BZ-9000 (BIOREVO) microscope for taking pictures of rat brain.
After That i have to merge these picture. But i found colored/dark square in final image .
How can I fix this issue?
Can anybody help me resolve the above issue, atleast give me some suggestion
Thanks
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I’m sorry, I didn’t understand. Could you please elaborate it
, please?
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Ground truth means usually boundary or outline of a cell usually annotated by pathologist in histology specimens. I am looking for a database which contains liver steatosis (fat accumulation) images where few ground truth data annotated by pathologist is available.
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Follow some of the high impact articles.
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I used an IHC staining for Pan-cytokeratin to test for metastasis from breast to brain. I am not sure if the only cells found in brain that are stained brown-ish are the tumor cells from breast cancer that metastasized or could be any other type of cell already present in the brain?
Thanks!
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Sorry for the late response. I would like to correlate with H&E. Can you please share that? Thanks
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Dear colleagues,
I was wondering whether there are any ways or any softwares that I can use to analysis the muscle cross sectional area in my H&E histology images?
I tried to use ImageJ thresholding, unfortunately it does not work efficiently for me. Thus, I was wondering whether there are any currently established methods.
Thank you very much in advance.
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I also want to calculate muscle fibers CSA in H&E staining using imageJ. Thresholding cannot be used but manually it can be done and it will be time taking process. I want to know about how many and how the fibers are randomly selected in a sample field, how many sample fields will be needed and what should be the magnification?
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I need histological images of paranasal sinus and tooth eruption for our new " Oral Histology" book project. We will mention provider at acknowledgements and under the image. Please do not hesitate me to contact me for details. #histology
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NT Fascicle 11 Chapter 1.pdf (arppress.org)
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I have measured breast tumor size using different modalities CESM and MRI. I have also the size on Histology.
I have a table of paired Samples Test, and the Sig. is .037 MRI and .523 for CESM when compared with the gold standard of histology. What does this mean??
Help xx
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The output table shows the degrees of freedom (df). The table also already tells you which modalities specifically differ. Please see my previous comments.
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I have scanned ovarian H&E slides and need to go through them and mark all of the follicles. There are several types of follicle (primordial, primary, secondary, antral, etc). I want to be able to quickly go through them and put a specific mark that indicates what type of follicle it is. The mark should be an overlay (not a permanent mark on the image) so that the original image is not altered. I want to be able to quantify the number of each follicle type at the end and keep track of everything. Open-source is preferable but a low-cost paid program would also be acceptable. I have messed around with several python based programs but none seem to fit the bill. Does anyone have anything that has worked well for you in the past?
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Hi Trent, have you done it? Automated the process?
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Hello!!
I am doing histopathological analysis of kidney sections with PAS stain. I want to quantify the staining intensity of the sections. can anyone explain what is the correct protocol for using the color deconvolution tool in ImageJ software?
Thanks in advance,
Sreyasi!
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Sreyasi Das . Once you have segmented or thresholded the regions you want to measure, you just have to "Set Measurements" to include Mean gray value (this is mean intensity), or integrated density, and you can then measure these regions.
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I would create my plates (a group of histologic figures) in MS paint, then save in irfanview, where I resize and adjust to desired dpi. I couldn't group figures, label, adjust contrast, etc in irfanview. I thought there should be a software that does it all.
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Hi Davinson,
You might want to have a look at GIMP: https://www.gimp.org/
You can think of this as a free and open source Photoshop that runs on Windows, Mac and Linux. The website has a bunch of tutorials for image manipulation, setting up layers, adjusting DPI etc
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I am analyzing a batch of adipocyte tissues using Adiposoft which is a plugin for Fiji. The program is taking the area of the cells and giving me their Equivalent Diameters (D-eq). I read that "From the diameter, the average adipocyte volume and lipid content can be mathematically derived" (Galarraga et al. 2012).
Could someone tell me what formulas (and the definition of their variables) I can use to find the Lipid content and the volume of an Adipocyte using the D-eq and Area?
I also have the size of the image in microns and an estimate of how many cells are in the image based on what Adiposoft counted during its automated analysis.
Galarraga, M., Campión, J., Muñoz-Barrutia, A., Boqué, N., Moreno, H., Martínez, J. A., Milagro, F., & Ortiz-de-Solórzano, C. (2012). Adiposoft: automated software for the analysis of white adipose tissue cellularity in histological sections. Journal of lipid research, 53(12), 2791–2796. https://doi.org/10.1194/jlr.D023788
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Wait since its the equivalent diameter do I just calculate the equivalent volume using the volume of a sphere formula? -_-
V-eq=(1/6)*Pi*(D-eq)^3?
And is the equivalent volume then equal to the lipid content?
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How would you evaluate the glomerulosclerosis condition of thhese glomeruli (picture 1,2)?
These are glomeruli of rat kidneys. The rats were treated after administration of streptozotocin. Untreated rats had significantly hypertrophied glomeruli with eosinophilic deposits (picture 3). The size of glomeruli of the treated group was standard, however the majority of glomeruli had minimized or fast no Bowman´s space (picture 1,2) - is this actually a normal condition? Inspite of that, kidney function parameters of treated group were similar to the controll healthy group.
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These glomeruli look like they are within normal limits to me. These look like PAS stains, so you can't say that there are eosinophilic deposits (as this stain does not contain eosin). The basement membranes within glomeruli are staining for PAS (normal), but there do not appear to be significant increases in PAS-positive deposits, or clear loss of cellularity. You cannot conclude as to "hypertrophy" of glomeruli based on the diameter of a glomerulus on a histologic section, as this will depend on where the section goes through the glomerulus. I think you need to have your sections evaluated by a qualified pathologist.
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Hello, I am writing because in our lab we have been doing some histochemical stainings and thus using water-based mounting media such as Mowiol 4-88. We have been running into a number of difficulties in the mounting medium preparation starting from the powder (we have been following the standard protocol provided by the manufacturer, e.g. https://www.polysciences.com/media/pdf/technical-data-sheets/TDS-20777.pdf), but we struggle to get the powder in solution and the end product is way too watery to be used for mounting (we have resolved to use much less water/buffer than advised, but it's still not great). We have also encountered precipitation issues when keeping the Mowiol mounting medium stock at RT for some time.
Does anyone have any protocols/tips and tricks to share to prepare Mowiol 4-88 mounting medium or any analogous water-based mounting medium? Thank you so much!
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I recommend using the glycerogelatin (ready to use) from Sigma. It is easy to open a slide using warm water.
Regards,
@
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Hello everyone.
Recently I got these images from a small project I'm doing, but as you can see, there seems to be some things that resemble "crystals" in my samples, of course they only appear in the experimental group. I've tried looking for "lipid droplets", "inclusion bodies", "crystals", "protein accumulation", but so far I haven't been able to find something that resembles what I'm seeing here.
Can someone please give me a hand in trying to explain what is it that appears in these samples?
Thanks for your time and kindness.
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Elena Lebedeva Good afternoon! the samples were sent to a histopathological laboratory, the magnification was 20X. I apologize for the low sharpness, it's a photo I took with my cellphone to the computer screen at that moment (I wasn't at my lab nor on my pc), my mistake. We considered the artifact option, but a histopathologist told us the characteristics matched with salt deposits, although we don't know how to prove this.
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I consider purchasing a SLEE automatic microtome, but I never had an experience with it. Can anyone here give an opinion on the instrument and on their service?
Thanks a lot
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unless , no
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Hello,
I work with a mouse model that in response to ultraviolet radiation develop cutaneous squamous cell carcinoma
To grade the tumours, I excise them from the skin on the mouse’s back and leave them in formalin to be embedded in paraffin. The paraffin and sectioning part is done by our experienced technician who has worked with this type of tissue before.
But when the tissue has been embedded in paraffin and left for a few days before sectioning, the blocks started to look wrong (see attached picture). There are cracks in the paraffin and in some places (picture left) it almost looks like the tissue is rejecting/repelling the paraffin.
Nothing about the protocol has changed, we do a overnight program before the paraffin embedding, and the only thing that has varied (a bit, if any) is the increase in tumour size. The tissue in formalin feels like it should, and others have used the same program as us with no problems.
Our working hypothesis is that it might be the temperaure of the cooling plate where the slide are placed after the paraffin. On the last batch, we used -14 C and still observed cracks in the paraffin.
Does anyone have experience with this problem or know what it may be?
Thank you in advance.
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Hello Celina,
here my embedding protocol. We use it for rat and mouse brains. Rat brains were dividet in cerebellum, frontal part and middel part so that you have tissue blocks not much bigger than 10 mm. After rinsing in buffer or tap water: 70% EtOH I -- 1 hour; 70% EtOH II over night (manual embedding) or 1 h in the embedding mashine; 100% EtOH I -- 1 h; 100% EtOH II -- 1 h; Isopropanol I -- 1 h; Isopropanol II -- 1 h ; Xylene (Tissue Clear) I --45 min; Xylene II -- 45 min; Xylen/Paraffin (2 part Xylene + 1 part paraffin; sorry I have changed it) -- 45 min at 60°C (Paraffin melting point 56°C); Paraffin I -- 1 hour at 60°C; Paraffin II overnight at 60°C, Paraffin III -- 3 hour at 60 °C. Blocking: I know the problem when you try to optimize the position of your block, epecially when you try to keep the tissue in a vertical position. This should be done as fast as possible to prevent that a thin paraffin layer which coveres the tissue, does not connect tissue and paraffin properly. Just in case it will happen then you should melt the paraffin again.
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I've been cryo-sectioning Xenopus oocytes and find these large internal branching structures in all of them (see attached image). I haven't been able to find a reference to it in the literature. I was tempted to say that it was ER, but I don't think it is. Does anyone have information on what this structure is?
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Hello Andrew Gipson,
In my opinion, it could be heterogeneous distribution of yolk platelets in the vegetal pole of Xenopus laevis oocytes. Two types of primordial yolk platelets (I and II) have been discriminated. After membrane fusion these precursors can be completely incorporated into the main body of existing platelets, numerous yolk crystals then merge and form one uniformly stratified core.
The yolk platelets ofXenopus laevis have been studied by thin-section and freeze-fracture electron microscopy to characterize the boundary membrane during yolk formation. Lipid droplets are tightly attached to the membrane at all developmental stages of yolk platelets. A direct connection of endoplasmic reticulum to the membranes of yolk platelets has not been observed. The proteinaceous yolk platelets tend to fracture along their periphery through the superficial layers.
For more detail see the following link:
Yolk organelles and their membranes during vitellogenesis ofXenopus oocytes. Roux's archives of developmental biology 198, 92–102 (1989).
Fluorescence lifetime imaging reveals heterogeneous functional distribution of eGFP expressed in Xenopus oocytes.
In this paper please see the figure 1B, similar to your attached image.
Best
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Occasionally when handling wetted picric acid or saturated solutions some solution can be transferred to gloves or a very small spillage (a few drops) might need wiping up with a tissue. I then dispose of these gloves and tissues in the bin where they can dry out. How dangerous are these dry residues in terms of explosion? What is the best practice in this case? Thanks.
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contact with even a small amount can cause an allergic reaction with symptoms such as skin redness, itching, rash and swelling.
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Hi there,
can anyone recommend a protocol for staining the mouse gamma-delta TCR in formaldyhyde-fixed, paraffin-embedded tissue ideally for immunofluorescence?
Thanks a lot!
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Hi... what is the best histology protocol for rat tissue (kidney, liver, heart, spleen)?
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You can review papers and follow the the staining method preferred.
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I will stain the osteoblast cells we planted on our hydrogels with Alizarin on the 14th and 21st days.
In the protocols I found, 2 grams of Alizarin Red S is dissolved in 100 ml of distilled water to prepare the dye. Unfortunately, I have just Alizarin (Mordant Red 11, 97% Sigma: 122777) on hand. Is it possible for me to implement the same protocol with this?
I have tried dissolving it in both water and DPBS but the dye sinks to the bottom and I get a feculent orangeish liquid. As far as I know, Alizarin Red S is not this color. Please help if you have any tips or experience on this.
I also noticed that when I used Alizarin Red S before, there was a lot of dye residue and it was difficult to see specific stains. No matter how many times I wash, I cannot remove the dye from the gels. I would be glad if you help.
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Dear Uzair Hashmi , thank you for your recommendations. As I said before, both distilled water and PBS did not help me dissolve Alizarin. So I could not filter either.
I just want to point it out in case anyone has the same problem later on. The product Alizarin (Mordant Red 11, 97% Sigma: 122777) I mentioned above seems soluble in 1N ammonium hydroxide. After dissolving the dye with NH4OH, just adjust the pH to 4.1-4.3. It worked fine for me. (Credit: Aditi Mahajan)
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Hi all,
I'm trying to perform Masson trichrome on frozen tissue sections however my fuchsin staining is turning purple once I add methyl blue.
My protocol is as follows:
1. Fix in 4% PFA
2. Rinse in distilled water.
3. Stain nuclei using celestine blue-haemalum method.
4. Differentiate with 1% acid alcohol.
5. Wash in running tap water.
6. Stain with acid fuchsin solution for 5 minutes.
7. Rinse in distilled water.
8. Treat slides with phosphomolybdic acid solution for 15 minutes.
9. Drain and stain in methyl blue for 90 seconds.
10. Rinse in distilled water.
11. Treat in 1% acetic acid for 2 minutes.
12. Dehydrate through alcohol gradient and clear in xylene.
Any help would be appreciated.
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Good day, what type of tissues did you stain?
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Hi all
I am making histological slices of bones without decalcification. Normally I only clean the bones with several baths in boiling water with bleach, but I have not had the best results with this method. I wanted to know if anyone has been doing any additional methods to remove fat and organic remains from the bones. Any comment would be very helpful.
Thank you in advance!
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I think the protocol found in this article (in attached) may actually help you.
Best wishes,
Sabri
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It is kidney obtained from a rat with early diabetes mellitus type 1 (streptozotocin model).
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Hi
I think the eosinophilic material in Bowman's space is protein debris. I advise looking for signs of proteinuria such as hyaline casts. Indeed, occasionally within the renal tubules there appear to be small amounts of eosinophilic (proteinaceous) material...
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What is the best software for analyzing the histological pictures?
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Dear Dr. Arash
Image J is appropriate for analyzing the histological pictures. you can get it from below URL:
Also you can Run ImageJ in Browser. This option is new possibility about image J software.
yours truly
Please don't hesitate from contacting to me for more details about this question.
+989125032346
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I am running an IHC experiment on mouse brain tissue that has been virally infected and I am looking to track infection by using and optimizing antibodies that detect immune cells, such as astrocytes and microglia etc.. In this experiment, I am blocking with normal goat serum (NGS) in PBS prior to using a goat anti-ALDH1L1 that reacts with mouse as a primary, and a donkey anti-goat as a secondary. This is the second time I run this with the same conditions and I dont see signal - it seems like the secondary antibody is not binding to/ picking up anything (similar to my negative control under the microscope). I am sure that there is antigen is the tissue - I previously used rabbit anti-GFAP and the signal of astrocytes is very strong and visible, so I would expect very similar outcomes with anti-ALDH1L1. Could this be due to the use of a blocking serum from the same species and the primary antibody and not the secondary?
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You should avoid using blocking serum from the same host species as your primary antibody that is detected by a secondary antibody against the same species because immunoglobulins in the blocking serum will compete for the secondary binding.
In your case, you should avoid using goat serum for blocking if you are using a goat primary antibody with anti-goat secondary antibody.
Use serum that is from the same species as your secondary antibody. You could also use BSA for blocking.
Best.
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Im working with fresh apical stems and this organ is very soft. After embed in Cryomatrix or OCT, it is posible to obtain around 40% of sections with good morphology (even cambium cells). But in the other 60%, shrinkage happens specially in cambium cells. Troubles:
- Samples after dry suffer of cell shrinkage, specially the cambium cells
- Samples after ethanol wash (for laser microdissection), suffer an even more shrinkage.
How could I avoid shrinkage in my cells? 
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شكرا لكم
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Is there any technique associated to histology that allows to detect a mutation in a specific gene of a single cell of a certain tissue? (i dont need to know the type or detail of the mutation, only that there is one), assuming that the rest of the cells have a wild type gene
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الطفرات التي تحدث في الخلايا الجسدية تسمى طفرات جسدية، ولا يمكنها الانتقال إلى النسل عن طريق العمليات التكاثرية في الحيوانات.
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Hi, I would like to find a good anterograde tracer. Which is better, BDA or Fluoro-Ruby? Thanks a lot in advance!
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I am using snap frozen muscle biopsies and am using a non-commercial antibody currently but would like to start buying a commercial antibody. 
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يمكن مراجعة المختصين
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For training a network I'm searching for a dataset of complex bio-images. I have seen some datasets for cell-classification, but these are usually simply amplitude-only samples, like these: http://imagej.net/Public_data_sets
Does anybody know any source of microscopic phase-images or does anybody have a set of complex images which could be used as a base?
I acquiered a small set using the quantitative Differential Phase Contrast Method provided by Lei Tian. These images are working quiet well :-)
Many thanks in advance!
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تجدها على النت يمكنك البحث
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I am trying to find any literature regarding the effects of diabetes on the amount of living PBMCs. Specifically using flow cytometry. In my own research i'm finding a difference between control and hyperglycemic groups.
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In tissue processing, xylene is toxic and difficult to regulate end point (especially manual processing). mineral oil - isopropanol combination can be used effectively. but what processor uses this method? also can the mineral oil be recycled?
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المعالج الدقيق أو المجهري أو الصغري (بالإنجليزية: microprocessor)‏ (يرمز له أحيانا ب µP)، ... وبذلك يستخدم المعالج الدقيق بالعديد من الوظائف،
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Dear colleagues,
I am currently looking for used but minimally working microscopy, histology and cytology equipment. Is there anyone who wishes to get rid of old things? E.g. microtomes, tissue processors, microscopes, etc?
Thanks.
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I will stain heart tissue slices with TTC in order to measure the infarct area. I read that the age of the heart after death is important factor for validity of the test. I wonder how long can I store the heart slices before TTC staining? What is the optimal time to do TTC staining after collecting the heart samples? 
Thank you.
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لا اعلم يمكن سؤال المختص في ذلك