H&E - Science topic

Thank you for sharing the H&E images.

#1A: This appears to be a perivascular lymphocytic aggregation, not a metastatic tumor.

#1B: The structure seems to represent interstitial edema, likely due to fluid accumulation during tissue processing. It does not suggest a tumor or a classic processing artifact.

#2 – #4: These areas show focal lymphocytic infiltrates rather than metastatic tumor deposits.

There is no definitive histological evidence of metastatic carcinoma in the provided sections. To clarify the nature of these foci and confidently rule out micrometastases—especially in cases of treated or regressed tumors—I would recommend performing immunohistochemical staining with appropriate markers (e.g., cytokeratins such as AE1/AE3 or tumor-specific markers depending on the primary tumor type).

I would put the slides in a minus 20 degree environment for an hour or so to adapt the temperature. For HE I would fix the slides then in 4% NBF for 30 min or 35% Formaldehyd for 1 min at roomtemperature. Then proceed with HE.

For IHC I would airdry the slides at RT. Then fix in 4% NBF if possible etc.

Slides for fluorescence microscopy must not be dehydrated for preserving fluorochromes. They need an aqueous milieu.

Dehydration and clearing are the steps before mounting with acrylic - non watermiscable - medium.

Methyl green is a commonly used stain in histology, and while it is generally quite stable and binds strongly to DNA and proteins, it can be removed with the appropriate chemicals. Here is a general protocol for destaining methyl green from a slide:

Remember that some tissues may be more delicate than others, and over-destaining can lead to tissue damage. Always monitor the process and be prepared to stop if the tissue starts to show signs of damage.

You may use consecutive sections of the same block and have the tissue evaluated by an experienced pathologist to obtain the histology and select the areas for special transcriptomics. But you need to be aware that using a consecutive section might introduce issues due to section-to-section variability requiring proper integration if you aim for single cell analysis of morphology and transcriptome.

Merck DPX we r using

yes.it used for osteogenesis and live/dead cells

I think it depends on how close those sections are to each other. For example, are the nodules in one slide present in another?

I'd suggest analyzing all of the H&E slides you have from each sample. You can then use the total number or the average across the slides, however, I would advise against just using the highest number in one slide.

Hi @Alberto Daniel Saucedo-Campose

I wan to focus on the inflammatory indexes and interaction of stroma cells and T&B cells.

On the contrary, could you please proceed with a new section, this time adhering to the following steps:

In conclusion, it's crucial to recognize that a uniform lab procedure may not be suitable for all tissues; the brain, for instance, cannot be treated the same as other organs. The thickness of sections should vary, considering that brain tissue is less resilient compared to other tissues. Experience plays a significant role in optimizing and adjusting lab protocols. If you encounter challenges that cannot be rectified, as you're all thinking it's a fixation error, consider taking your block to a nearby histo lab. Their expertise can help produce a well-stained outcome. Since we cannot visually inspect the issue, our assistance is limited. But I doubt it's a fixation error!

However, unless you confirm challenges like brittleness or difficulty obtaining a section or ribbon during sectioning, I can tell you it's likely not a fixation error. Your nearby histo lab remains a valuable solution for resolving such issues.

Sincerely, your nearby histo lab is your solution arena, consider taking your block there for help...

Novel context-based segmentation algorithms for intelligent microscopy

Perhaps H&E/2 means: Deconvolution 2 of Stain mixtures....

Dear Lucas Vajko,

These granules seem to be parts of nuclear remnants of granulocytes (degenerative changes). Such changes are seen in mild inflammatory processes.

Best wishes

Please, send me a photo (photos).

Best,

Peter

Before you start with you staining fix the tissue sections in 4% PFA pH 7.4 over night in the fridge (4°C). This procedure protect the tiusse and kills the virus. After several rinses in distilled water you can start with your staining precess.

POU5F2

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.

Hi,

Well, post your image and explain the issue (the post is kind of old). I will try to help if I can,

Cheers,

J

Hi Monica,

I know skeletal samples (bones and teeth). I did use human bones for my study. I am pleased to share my little experience with you, hopefully, it is useful. Please consider some things such as:

1. Type of histological slides. Are they positive charges?

The ideal slides I mounted the sections on are HistoBond®+ adhesive microscope slides (Paul Marienfeld, Australia). Positive charges enhance the adhesion of sections on slides. It also supports subsequent steps such as staining and washing. Good adhesion prevents the loss of samples during histological steps.

2. The thickness of sections.

I did try a series of thicknesses such as 5 um, 7 um, 10 um, and 15 um. The best thickness for adhesion on (+) slides is 5 um. The more thickness, the more annoying troubles. It also depends on the choices of fixing, for example, FFPE samples can be easily performed with thick sections (up to 50 um), due to the very long decalcification (up to 6-8 weeks). Another example is MMA (methyl-methacrylate) Resin, the samples are difficult to be mounted on slides with thicker sections (7-15 um thickness).

3. Decalcification time.

It influences softening process. Skeletal samples always have problems related to decalcification. How long is enough for decalcifying a bone specimen? To ensure your samples are not over-decalcified, an X-Ray can access the ending point of mineralisation. Please consider a quick surface decalcification (it takes one to two hours) prior to sectioning. For example, my FFPE/Resin bone block was still hard to cut/section by microtome/specific machine. I put the bone block face directly on 0.5 M EDTA for 30 minutes to one hour or two hours in a small container. Then, leave it on ice and section it at a choice of thickness ( 5 um thickness is a priority).

4. Embedding medium/solution.

Prior to embedding, ensure your samples are decalcified/ calcium removed completely. You can check the ending point of decalcification by X-Ray as mentioned above.

In brief, there are three main steps prior to sectioning and staining including:

Fixation (10% Formalin or 70% Ethanol) > Decalcification (4 - 8 weeks, check by X-Ray) > Embedding (Paraffin or MMA resin) > sectioning > staining > analysis.

Hope it helps!

If you are interested in reading details of my technique, please see my study here:

https://doi.org/10.1007/s12024-021-00428-3.

Regards,

Good luck with your experiment.

Dear Mallu Abhiram Charan Tej - H&E staining can be used to quantify angiogenesis. You would need to ensure that the section size is identical between your experimental and control samples (in case you are performing a comparison).

Just a suggestion, but Mallory Trichrome staining might give you better clarity visually as the erythrocytes are stained a distinctive red in a blue-ish background.

Good day.

H&E is a routine stain and I am not sure that it can show the expression of one protein. May be you need IHC protocol?

اعطني قليلا من الوقت حتى اجد من يساعدك

شكرا جزيلا

يمكنك مراجعة البحوث الخاصة بالاساتذة للحصول على الجواب

These are the ganglionated plexuses of the heart which lie within the epicardial fat predominantly in the atria. You can also see the beginning of a nerve in "high zoom top.png". The individual cells are called ganglion cells. You can confirm using immunohistochemistry such as PGP9.5 or NSE.

María Martín-Grau I hope you find these answers helpful.

Ok Biagio I'll take this into consideration. Thanks so much for your help.

Hi Sara,

It would be helpful if you could elaborate a bit more about the kind of quantification you aim to do. Regardless, if you have not fully committed to it yet, I would recommend you check out some other tools that might be easier to work with.

For histology I found QuPath to be reasonably user friendly and more importanlty, it comes with good quality documentation for most basic analysis. If you follow the link, you will find information on how to install it (it’s free), tutorials on how to identify and measure areas, separate the haematoxylin and eosin channels and quantify cell numbers etc.

Hope this helps!

https://qupath.readthedocs.io/en/latest/docs/tutorials/index.html

I can really recommend you to start a collaboration with a pathologist at your clinic. We faced the same issue last year with HE stained brain tissue. The consulted neuropathologist could score tissue changes with amazing speed and high details. Don't hesitate to directly write experts, worst thing that can happen is that they don't reply and from my experience people are happy to help.

Hi,

The answer for you is yes, FIJI can do all the things you want.

I fear just if you really want an absolute number, you would need to go for fluorescence microscopy, stain with something like Hoechst and count nuclei. Its an easy to use and cheap stain.

In case confluency is enough to normalize your samples, most of the time it is if the cells don't grow to much on top of each other, you do not need the fluorescence staining. Best would be a Phase Contrast Image, highlights the cell boarders really well. Depending on your setup with a brightfield correction and smoothing of the image might be necessary before threshholding to generate the best result.

There are some algorithms available that try to seperate and count single cells out of a phase contrast image, from my experience it doesnt work too well.

If you need more help with FIJI i can send you some workflows.

Good Luck

Jürgen

Solutions:

3% Acetic Acid    Acetic acid    3.0 ml    Distilled water   97.0 ml    Stir together. Stable at room temperature for months.

Alcian Blue, pH 2.5    Alcian blue   1.0 g    3% acetic acid   100.0 ml    

Thymol crystals   Dissolve alcian blue in acetic acid. Check pH; adjust the pH as needed using acetic acid to pH 2.5.    Add a few crystals of thymol to prevent mold growth. Solution is stable at room temperature for    months and may be reused until weak.

Mayer Hematoxylin: Commercially made

0.25% Hydrochloric Acid     Hydrochloric acid     2.5 ml     Distilled water     997.5 ml     Carefully add hydrochloric acid to the distilled water slowly. Stable at room temperature for months.

0.25% Ammonia Water     Ammonium hydroxide     1.0 ml     Distilled water     250.0 ml     Slowly add ammonium hydroxide to distilled water. Use for one day only.

Eosin: Commercially made

0.25% Metanil Yellow     Metanil yellow     0.25 g     Distilled water     100.0 ml     Glacial acetic acid     0.25 ml     Mix together well. Stable at room temperature for up to one year.

Method:

1.   Deparaffinize and bring sections to water

2.   Stain with Alcian Blue, pH 2.5 solution15 minutes

3.   Wash well with water

4.   Stain in Mayer Hematoxylin *4 minutes

5.  Rinse in running water, several changes

6.   Differentiate in 0.25% hydrochloric acid 2-3 second

7.   Rinse in running water, several changes

8.   Blue in 0.25% ammonia water2 -3 seconds

9.  Rinse well in running water, several changes

10.   Place in 70% ethanol1 minute

11.  Stain with Eosin Solution1 minute

12.  Dehydrate in 95% ethanol30-60 seconds

13.  Dehydrate in 100% ethanol, two changes30 seconds each

14.  Place in Metanil Yellow solution**1 minute

15.  Rinse with ethanol, 2 changes10 dips each

16.  Clear with xylene, 3 changes2 minutes each

17.  Mount in a resinous medium

*For automated stainers, run a program on the stainer that takes the slides from water, through your routine H&E and stops at the second change of absolute ethanol.

** Timing of the Metanil Yellow is critical. If stained for too long, increased background staining will occur.

Results: Nuclei - blue Cytoplasm - pink-red Mucin – Turquoise for Barrett’s Esophagus Goblet Cells (some gastric mucin will stain a faint blue) Collagen - yellow Smooth muscle - salmon

Have fun :)

Hi, I think this web site will be helpful for you

Thank you Dr Ogunlade Babatunde for your answer. It is much appreciated.

Regards.

Sunday

Thanks for your reply Fred Sinowatz . Which fixative would you recommend then?

You can try

1. Carnoy's solution for fixation of myxamoebae, then stain with 2% Gurr's acetoorcein in 45% acetic acid following hydration through alcohol series and hydrolysis in 1N HCl (Sussman R, 1961).

2. Fix the cells in methanol/glacial acetic acid (3:1, v/v), then stain with 10% Gurr's giemsa stain in Sorenson's phosphate buffer (ph-6.8) (Brody and Williams, 1974).

These methods will be helpfull when you want to stain nuclei of vegetative myxamoebae. Hope it helps.

Following

It depends on what you would like to measure. To separate H and E dyes to quantify them separately you can use colour deconvolution: https://imagej.net/Colour_Deconvolution

To estimate e.g. cells number - you can use one of the plugins to do this after deconvolution/thresholding (but for cell counting I will strongly recommend stereology), to measure areas - you can outline your ROI by hand and measure it using Fiji, you can also use threshold and binarize the picture to measure its features etc, etc, there are many possibilities, depending on what should be measured and how the tissue looks like.

Aarifa Nazmeen you can in-cooperate the steps of IHC into H&E protocol for better results.

Tanya Abo-Shaban You may consider following to prevent your tissues from falling off the slide during staining:

1. Use a good quality and adequate amount of mounting material (too little or too much mounting material will let your tissue section fall off during staining) on your non treated glass slide before placing a tissue section or you can always buy coated glass slides for mounting you sections.

2. The thickness of the section should range between 3-5 micron meters for better staining results.

3. Mounted tissue sections can be left for heating at 37C overnight or 40-45C for 2 hours before staining. Over/ prolong heating can cause your tissues to dry/ burn off and eventually fall off from your slide even before staining.

Best of luck

Thank you so much!

This might an issue, however I also understand your trying to keep your staining process on the lowest price as possible.

I believe coating with Poly-L-Lysine might help to solve your problem, and this is still cheeper then coated RTU slides.

Dear Matthew,

since DAPI is a nuclear dye, it is not sufficient to identify HCC cells. If you want to identify such cells in your sample you can co-localize DAPI with Squamous cell carcinoma antigen (SCCA).

Good luck with your research,

Davide

This is a tough one. The contrast of the nuclei relative to the background is not good, making hard for automated thresholding. An image is attached of my effort. It took a lot of processing and the result is still not all that good. I used the macro recorder to note the steps. I converted it into an 8 bit image, inverted it (ImageJ is set to work with fluorescence where the signal is brighter), subtracted background, smoothed it, used auto thresholding with default algorithm to create binary, used binary filters (fill holes and watershed) to fill holes and separate boundries, then analyzed particles with size and shape criteria of the binary to create ROIs. run("RGB Color"); run("8-bit"); run("Invert"); run("Subtract...", "value=125"); run("Gaussian Blur...", "sigma=9"); setAutoThreshold("Default dark"); //run("Threshold..."); //setThreshold(18, 255); setOption("BlackBackground", true); run("Convert to Mask"); run("Fill Holes"); run("Watershed"); run("Analyze Particles...", "size=400-Infinity pixel circularity=0.25-1.00 exclude clear add");

Pancharatna Katti Hi and thanks. The logic for thickness was, the thinner the sections, the quicker they dry. 10-20um is common for frozen sections but not paraffin, which was why I scaled down to 3-5um. Did not see a difference and I still struggle with the problem, I have done some troubleshooting by increasing length of times for ethanol dehydration (1 minute each, 70%, 90%, 100%, 100%) and clearing with a xylene substitute, even though the mounting media contains toluene, results are the same. I will be storing some samples for paraffin until I eventually (if ever) get the frozen sections to work.

Dear Ghulam

You could find an authorized and easy technique with:

01‏/10‏/2012 - Bancroft's Theory and Practice of Histological Techniques E-Book. 7th Edition. Authors: Kim Suvarna. eBook ISBN: 9780702050329.

Good luck

Dear colleague

In difficult cases we can use bcl-2 (diffuse staining in bcc, only basal layer staining in trichoepithelioma), CD34 (positive in peritumoral fibroblasts of trichepithelioma not bcc), CD10 (positive as epithelial expression in bcc and stromal expression in trichoepithelioma) and stromlysin-3 (positive in fibroblasts of bcc not trichepithlioma). Refer to Weedon's textbook of skin pathology or other dermatopathology references.

[Apologies for lengthiness]

Cara Maya (guessing you are female):

You might have further reading to get completely led astray…:

Your handling and doing always depends what your „real“ target with the PMMBC's is:

histo-morphological proof ONLY, further processing of the cells from slide (e.g.collecting RNA, try ultratsructural analysis later on), etc., etc.

And, as always, one has to find the most promising protocol out of many, sometimes by trial and error…(;-().

It may be of help to you to read / go through:

https://www.researchgate.net/post/How_can_I_perform_hematoxylin_and_eosin_H_E_staining_of_non-adherent_cells

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http://cmurdc.cmu.edu.tw/HVIS/5-introduction/LMPC/A1-SPECIMEN%20PREPARATION%20PROTOCOL.pdf

Preparation of slides - Mounting sections onto slides - Staining of the sections

(quote p.-6-):

Cytospins can be prepared on glass slides or on membrane slides. After centrifugation with a cytocentrifuge let the cells air-dry for 5 minutes. Then fix for 5 minutes in 100% methanol.

Allow the cytospins to dry at room temperature before staining.

Distribute a drop of (peripheral) blood or material of a swab smear over the slide.

Let smears shortly air-dry and fix them for 2 up to 5 minutes in 70% ethanol.

and

(quote p.-7-):

HE-staining is used routinely in most histological laboratories and does not interfere with DNA and RNA preparation. The nuclei are stained blue, the cytoplasm pink/red.

10 minutes Mayer’s hematoxylin solution

10 minutes rinsing in running tap water

5 minutes Eosin Y

then: increasing Ethanol series

[and finally:] let air-dry [NB: nothing said about handling for permanent slide]

3 minutes Mayer’s hematoxylin solution

3 minutes rinsing in RNase-free tap water

30 seconds Eosin Y

then: increasing Ethanol series

[and finally:] let air-dry [NB: nothing said about handling for permanent slide]

The nuclei are stained dark blue.

5-10 minutes Methylene Blue solution (0.05% in water; SIGMA, #31911-2)

rinsing in Aqua dest

[and finally:] let air-dry at roomtemperature [NB: nothing said about handling for permanent slide]

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Quote from Current Protocols in Molecular Biology Online Rapidly fix cytospins (cf: http://www.geguchadze.com/PDF/protocols/CPonline/Doc/19590-19590.html )

[ 7. When the alarm signaling the end of the spin sounds, quickly remove the assembled collection chambers. Open the chambers and remove the slides by lifting the blotter away from the slide ] This method avoids damage of cell membranes and thus smearing. 8. Quickly transfer slide into 95% ethanol without allowing the specimen to dry. Fix 10 min. Transfer slide to 70% ethanol for 30 sec. 9. Proceed to H&E staining (see Basic Protocol 5 = @ http://www.geguchadze.com/PDF/protocols/CPonline/Doc/19592-19592.html ) or other stain of choice. Taken from: From Current Protocols in Molecular Biology Online Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.

(NB: there also the problematic instruction to centrifuge/spin down the cells with „1500 rpm“…see *) NB below… It might be right statement when using a "cytospin(R)". For me such a high rpm number points to the g-force I preferably would use: in the range of ~approx. 900g - 1000g - 1100 g)

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HU et al. 2015: Research Article

Xiaotang HU, Verronika Laguerre, Daniel Packert, Alice Nakasone, and Lynn Moscinski

International Journal of Cell Biology Volume 2015, Article ID 813216, 4 pages

@ https://www.hindawi.com/journals/ijcb/2015/813216/

(quote from that article):

2. Materials and Methods

2.1. Preparation of Slides and Glass Spreader

„…..cells were harvested into a 15 mL polypropylene centrifuge tube and spun down for 8 min at 600 RPM (80 g)*), after which the supernatant was discarded and the cells were resuspended in 0.5 mL of culture medium and 1-2 drops of the cell suspension were placed on a slide in the central area and moved around to form a thin and even film with a glass spreader. The glass spreader was made from glass transfer pipette over an alcohol burner for few seconds to minutes.“

2.2. Cell Staining

Two types of stain methods were used in this study: Giemsa and methylene blue/eosin. For methylene blue/eosin staining, the slides were fixed by placing 2 drops of fixing solutions on the slides and air dried in a biological hood (2 min) or on bench (3–5 min). Next, two drops of methylene blue (1%) were added to the slides and left on for 2 min, after which the excess stains were removed by placing a piece of paper towel on the stain briefly (1 second). Subsequently, two drops of 1% eosin were added to the slides and incubated for 2 min at room temperature. The slide was rinsed briefly with small amounts of tap water, after which one small drop of mounting medium was added to the slide and covered with a coverslip. For Giemsa staining, 2 drops of 5% Giemsa were added to a fixed slide followed by the procedure described for methylene blue, omitting the steps for second stain. In order to test whether the slides prepared by this method are good for commercial stain kit, we also used the DIPP KWIK Differential Stain Kit (American MasterTech, Lodi, CA). [end of quote]

NB: *) the mentioned processing data for the cell-centrifugation, namely „600 RPM (rotations per min) (80g) without mentioning the type of centrifuge and rotor is misleading and therefore useless (applying „80g“ to me seem to be a really low force to collect the whole cells on bottom of the tube….)

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www.uab.edu/sites/luckielab/protocols/cytostdt.doc;

www.ihcworld.com/histology.htm;

https://pdfs.semanticscholar.org/12bb/cbe7243a534de3ff63cfeb08de6998a62eea.pdf;

https://books.google.at/books?id=fjouSV2R_fEC&pg=PA105&lpg=PA105&dq=Cytospin+preparation+and+H%26E+staining+protocol&source=bl&ots=SgD7aKc7rR&sig=uegkD0wY1Kn2elzg0nxYDkRedFI&hl=de&sa=X&ved=2ahUKEwjI8eTa3P7eAhVmkosKHePcC6w4ChDoATAKegQIARAB#v=onepage&q=Cytospin%20preparation%20and%20H%26E%20staining%20protocol&f=false;

https://books.google.at/books?id=l4IgKRRJegsC&pg=PA312&lpg=PA312&dq=Cytospin+preparation+and+H%26E+staining+protocol&source=bl&ots=Lk2XkEQBmg&sig=RMH3mhH9xpVsA-cgpHmOL8BclCs&hl=de&sa=X&ved=2ahUKEwjI8eTa3P7eAhVmkosKHePcC6w4ChDoATAMegQIABAB#v=onepage&q=Cytospin%20preparation%20and%20H%26E%20staining%20protocol&f=false;

http://screening.iarc.fr/doc/Cancer_resource_Manual_3_Cytology_New.pdf (see chapter 4 and chapter 5, respectively);

and , last but not least:

Comparison of FNAC smears, cytospin smears, and cellblocks of transthoracic guided FNAC of suspected lung tumor: A study of 100 case, by Ankur Singh Kshatriya, Pravina M Santwani, 2016

(J.Cytology, unfortunately only PPV-pay per view: @

http://www.jcytol.org/article.asp?issn=0970-9371;year=2016;volume=33;issue=3;spage=141;epage=144;aulast=Kshatriya).

Cordiali saluti, W.H.M

Hi Mohamed Mahdy ,

Thank you very much for sharing the protocols. I´ve tried the protocol for Acian blue - nuclear fast red as above. The staining is fine, but I faced another problem: as I used Kawamoto film for sectioning of undecalcified tissue, final clearing step with xylol made the glue from the film melted and messy.

Would you have any suggestions for this issue?

I think you could have some hair contaminating these tissue samples. The regular striations could be pigment bodies.

Since the formalin reduce the natural color of the preserved specimen, glycerin enhances the colour of the specimens in the long term display and used in Kaiserling solution

With acid phosphatase you can identify macrophage

With Alkaline phosphatase you can identify neutrophil

To see neuron/glia in hippocampus, Niissl stain (Cresyl voilet) is the conventional one.

Dear Roman, interesting aspect..."H&E is not suitable" (because of your task or because you don't get optimal staining when trying H&E?).

It would be interesting to know about the source of your MMA (which one?, which supplier, mixture, hardener/accelerator, etc.,etc.)

I'd guess:

Toluidineblue (at an acid, as well as basic/alkaline pH)

other cationic dyes in distilled water, Safranin O- red with methyl green counterstain, Von Kossa, GOLDNER or Masson trichrome, etc....( most stains will work with sections made of MMA plastic ).

Generally, I'd like to cf. to ResearchGate archives:

If you have available: check: BANCROFT/STEVENS:

Theory and Practice of Histological Techniques

the paper of ERBEN 1997:

(= free access pdf see[prior to paste the URL into your browser delet the under-line-character added inbetween http_ and _:/journals.sagepub.com ...... / !)

http_://journals.sagepub.com/doi/pdf/10.1177/002215549704500215

Keklikoglu N, Akinci S.

Comparison of three different techniques for histological tooth preparation. in Folia Histochem Cytobiol. 2013;51(4):286-91. doi: 10.5603/FHC.2013.0039. [will not solve your problem, I know, but perhaps interesting to compare. @ https://www.ncbi.nlm.nih.gov/pubmed/24497133; pdf can be found @ https://pdfs.semanticscholar.org/68ae/ca002b8a3dbe3f1b22c40d46f4145e9bbafb.pdf

Perhaps will come back later on with solutions...

I would take alternated sections (1 for CV, 1 for H&E). Mount your sections either on chromalum gelatine coated slides (after air drying overnight you can stain the slides next day) or on Superfrost plus (you have to dry them at 37°C for minimum 5 days, otherwise your sections will float away during the staining process, that's my expierence). Rehydrate the sections in 2x3 min in Xylene, 1x3 min Isopropanol, 2 x 3 min 100% Ethanol, 1x3 min 70% Ethanol, short rinse in destilled water , staining process (CV 3min or H&E duration depends on your protocol), dehydration and diferentiation in anscending alcohols, clearing in Xylene, mounting in Depex. Now you can take photo images, count cells or nuclei, what ever you want. Hope, this short instruction will help you.

Dark-Field microscopy may assist with identifying positive cell but it may be difficult to identify where the cells are specifically in the tissue.

Regards

Chris

Hi Doris,

From my own experience, though not ideal, it is possible to make out the cortical layers on the basis of their respective cellular components, density etc. However, you are correct that CFV (for Nissl substance) or NeuN (IHC) is far more appropriate for this purpose.

Hope this helps.

Ahmad

Dear Muzahir, goblet cells have various functions. One of them is making a resistant layer lining the intestinal mucosa. It seems the number of these cells affects the normal and natural resistance of intestinal wall against pathogens.

When the sections are sectioned and with scatters, we rinse the paraffin blocks in 1% ammonium hydroxide in distilled or tap water for 30s or a minute. This solution hydrates the sample and the next sections will be good. A second tip is using silanized glass slides to avoid tissue loss.

Dear Neda,

in my experience it works best to fix your rat skin samples in Bouin´s fluid (12hours) transfer them to 70 ethanol and than embed them in a conventional manner in paraffin. Usually you do not get good results for IHX from cryosections of rat skin.

Rgards, Fred

Hello again,

on the left side it is surely lymphatic tisse and in the middle it looks like pancreatic tisse (exokrine part), because it has the morphology of an albuminous gland. I remember that in mice and rats the pancreas is embedded in the omentum majus or the mesos of the intestine and is not a organ of itself like in humans. Therefore I'm not wondering that you will find such tissue in your histology slices.

Sincerely Yours

Stefan

EDTA decalcification method could protect the integrity of mRNA in mouse tibialis, and preserve the tissue structure for the histological section to determine gene expression, immuno- and in situ histology. Please read a related reference as follow:

Daniele Belluoccio, Lynn Rowley, Christopher B. Little, et al (2013) PLoS One 8 (3): e58154 doi: 10.1371/journal.pone.0058154

Tateki Kikuchi

I feel that it will be a problem to cut Daphnia on microtome. Maybe you can use our methodology which was prepared for rotifers:

Best regards, Ania

Eosin is a fluorochrome and it leaks out in alcoholic and aequous solutions. It would be recommendable to destain the slide before IF in acid-ethanol. I think hematoxylin is the minor problem.

Do gradual sucrose incubations, for example, 10, 20 and then 30%.

Make sure your tiny amount of acetone remaining will degrade your antibody. needs good washes.

Finally, make sure you are blocking remaining active aldehyde groups with 0.23% NH4Cl or 0.1N glycine.

Test first whether you can show any positive signal in whole mount in situ antibody staining (WISAS) by adopting every chemical use as in Cryostat.

Good luck!!!

This depends on the temperature in your oven. If only the paraffin melted it should be okay still. I would stain them and have a look under the microscope.

My samples are mice footpad. I have attached a picture for your reference. After infection,  the footpad gets a little swollen. So we want to check the presence of bacteria as well as check cell infiltration by histology. (we also to FACs). 

We have never performed histology of this region in our lab. In literature I find few only fixing and few happened to fix and decalcify before making sections. 

If I have to decalcify? can you tell me how long and what is preferred to decalcify? 

Thanks for your response

Dear Gudrun,

Thanks for the information. I am sorry I didn't mention I need it for paraffin slides. OK, now I know why they don't have a small capacity one....

Thank you so much.

Hi Lien!

Neurodegeneration is a multistage process, which depends on various factors. First of all, you have to decide what a model you want to use and what kind of cellular changes you want to see. Neurodegeneration may start from peripheral parts of neuron or may be initiated in the body. One more important question is how long it takes to develop neurodegenerative changes. When you will be able to answer these questions for yourself you can start to choose appropriate IHC or IF methods of detection.  

Thanks for all the suggestion. I think I will need to do some other things (such as EM) first to narrow it down in order to do DNA test...

I think the protocol looks ok. Also the use of reagent alcohol (ethanol+methanol+isopropanol) should be ok, as fas as it is water-free in the 100% alcohol. 8 hours in sum in high-percent alcohol seems rather long for small brain-samples, but should not result in insufficient dehydration.

Is the surface of the paraffinblock "wet", slushy and does it sink in the center until the next day? These would be signs of residual xylen or ethanol in the tissue. Then one can put the tissue again in molten paraffin for an additional day and then cut again.

Check, if the reagens in the processor are fresh and in the right order.

I'd like to suggest the use of Gendre Fluid (alcohol 95-96% with Picric Acid diluted until saturation+Acetic Acid+Formalin) (=Alcoholic Bouin) fixing by 3 days (maximum). Epithelial (glandular) tissues are very delicate and Gendre fluid will fix the tissue fast. This fixing will permit you to develop PAS protocol for glycogen too, and the tradictional H-E.

Literature: http://pt.slideshare.net/ahmededrissi1/routine-histological-techniques

Best regards.

Completely agreed with Gudrun.

Cheers,

Ivan

I am so sorry that I delayed for one month. 

I will try to use cryoprotection and use liquid nitrogen to freeze the samples next time.

Thank you so much.

Just as earlier mentioned, thawing will dissolve the ice crystals and affect the cellular intergrity and morphology. If tissues were frozen previously in glycerol, this scenario may have been avoided as it prevents the crystallization of ice within the cells. Cryosectioning is more appropriate as Mahdy memtioned.

Good luck.