Histology - Science topic

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.

Please, send me a photo (photos).

Best,

Peter

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

pH. of ovaries provide protection against various microorganism. Beside that ,hormones provide nourishment to ovarian cells. Egg is released from ovaries about three months

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

Dear Sir,

The attached paper might be helpful for you.

I will do on two adjacent sections, on for histology and other for IHC. that will be better

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

Thank you so much, it could be some debris and air bubbles, as well as an adherent of gelatine particles

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.

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.

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.

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.

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.

Thanks for replying, do you have any refrence for that scoring?

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.

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.

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.

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.

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.

Pure ethanol will evaporate if you leave the tube open. It does take a while though.

Hello Jessica,

can you give some more details about the staining protocol and how you have prepared the staining solution?

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!

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.

I have paper about this in my page

Hi Chahrazed Kaoudoune

Please find the attached links, you can check the histopathological and anatomical differences between human and rat liver.

1. https://focusontoxpath.com/comparative-histomorphological-review-of-rat-and-human-hepatocellular-proliferative-lesions/

2.

Thanks

Samir Ranjan

Dear Geir Bjorklund, Duc M. Hoang, John Hildyard, thank you very much for your answers and recommendations!

There could be a number of reasons for weak or absent signals in immunohistochemistry, including:

thank you for sharing Dr. Juli, I think you can use the same protocol for dehydration of other tissues

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.

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.

Barbara Loi Omar Hernando Avila-Poveda (Avila-Poveda OH)

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!

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):

Summenformel: C20H19N3Na2O9S3

Molekulargewicht (g/mol)

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.

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

Hi Yifu Jin

*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

Try using Histochoice products (clearing and adhesive).

Hi Anna,

if you have protocols of the staining procedures and recipes how to prepare the dye solutions then it is not so difficult.

https://www.medimops.de/kiernan-j-a-histological-and-histochemical-methods-theory-and-practice-taschenbuch-M00750649364.html. If you need protocols for your stainings the book-link above could help you.

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.

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.

String theory.

Wonder how many new posters can offer the same information already posted numerous times.

I’m sorry, I didn’t understand. Could you please elaborate it

, please?

Sorry for the late response. I would like to correlate with H&E. Can you please share that? Thanks

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?

NT Fascicle 11 Chapter 1.pdf (arppress.org)

The output table shows the degrees of freedom (df). The table also already tells you which modalities specifically differ. Please see my previous comments.

Hi Trent, have you done it? Automated the process?

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.

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

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?

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.

I recommend using the glycerogelatin (ready to use) from Sigma. It is easy to open a slide using warm water.

Regards,

@

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.

unless , no

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.

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

contact with even a small amount can cause an allergic reaction with symptoms such as skin redness, itching, rash and swelling.

this website may be useful

You can review papers and follow the the staining method preferred.

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)

Good day, what type of tissues did you stain?

Dear Dr Maria Eugenia Pereyra,

I think the protocol found in this article (in attached) may actually help you.

Best wishes,

Sabri

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...

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

Hello Karim Zedan

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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