Questions related to Histology
Although the donut concept of the optic disc is widely published in glaucoma but it is not supported by any histology in the textbooks.
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?
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.
What type of study (or studies) could prove or disprove the hypothesis of parthenocarpy in the Cannabis L. plant genus?
Hey everyone, has anyone ever performed immunohistochemical staining followed by histochemical staining on the same histological section (like a double labeling)?
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!
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 ?
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!
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
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.
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?
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?
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.
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 :-)
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.
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?
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?
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!
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
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.
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.
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
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)
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.
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
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)
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.
Thanks in advance.
I would like to know whether the following compounds can be used interchangeably for Van Gieson staining:
Acid fuchsin: https://pubchem.ncbi.nlm.nih.gov/compound/Acid-fuchsin
Acid fuchsin calcium salt: https://pubchem.ncbi.nlm.nih.gov/compound/Acid-Fuchsin-calcium-salt
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?
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.
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.
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?
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...
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
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.
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?
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.
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
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??
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?
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,
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.
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
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.
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!
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.
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
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.
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?
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.
can anyone recommend a protocol for staining the mouse gamma-delta TCR in formaldyhyde-fixed, paraffin-embedded tissue ideally for immunofluorescence?
Thanks a lot!
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.
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.
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!
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?
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?
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
I am using snap frozen muscle biopsies and am using a non-commercial antibody currently but would like to start buying a commercial antibody.
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!
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.
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?
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?