The H&E Staining Protocol
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- เผยแพร่เมื่อ 30 มิ.ย. 2024
- A first person view of how to manually stain slides using the H&E staining method.
The protocol demonstrates use of Ehrlich's hematoxylin recipe which is designed to be used regressively (i.e. overstraining following by a differentiation step).
Excellent content! Thanks a lot!
Thank you Sir for this kind of video. It really help for me to pass my subject. Godbless to you
You are most welcome Ira.
Thank you for your effort. This is really a great video
Many thanks!
Hello, what are the most widely used fixative in HandE staining method
A. Formaldehyde
B.zenker formol
C. Picric acid
D. Osmic acid
Please help me answer
Wonderful Video. May I ask why did you overstain?
Interesting 👌...I have a question..what is the type of camera you are using for the point of view camera view? I could not find a very good quality camera resolution that can record videos. Thank you so much for your videos🙌
I use a GoPro camera mounted using a head strap.
I enjoy your tutorial
Thanks Kawiso!
Let me know if you have any questions.
Damien
Very informative thanks a lot
My pleasure
These are great :)
Hello, what are the most widely used fixative in HandE staining method
A. Formaldehyde
B.zenker formol
C. Picric acid
D. Osmic acid
Please help me answer
Thank you so much.
You're welcome!
Thank you for the video. Are those frozen sections or paraffin section?
Hi Yuganthini. Paraffin sections are used in this demonstration.
Will have to try this with my pulmonary tissue, I’m having a difficult time with the protocol I have now. What dilution are you making for the ammonia and the acid alcohol?
Hi Benny, There are multiple factors that might be causing you problems. I would suggest checking the level of staining with hematoxylin immediately after rinsing off the stain in water and treating the slide with dilute ammonia. If the level of nuclear staining is poor, then your hematoxylin solution may be the problem, or you may just have to stain for longer. Depends upon what recipe of hematoxylin that you are using. In any case, our dilute ammonia is prepared by adding 1 mL of ammonia to 999 mL of deionised water and the acid alcohol is prepared by adding 5 mL of concentrated hydrochloric acid (38% w/v) to 995 mL of 70% ethanol (v/v) in deionised water. Good luck and let me know how things work out!
@@damienharkin thank you so much! I’m a grad student but unfortunately don’t have much support in terms of my histology work. I’m very grateful for your advice! I will let you know how things progress. :)
@@damienharkin I tried your recommendations and the tissues look great until the last xylene step for dehydration at the end. When xylene is wet and on the slide, under the scope the tissues look perfect but when the xylene dries, the tissue turns extremely dark and all demarcation between cells is lost. I’ve been playing around all day to troubleshoot but have not had any luck. Any thoughts would be greatly appreciated!
@@damienharkin I’m wondering if I need to use my permount and coverslide immediately after I take the slide out of the xylene? I was told the slide needs to dry before I use the permount but by then, the tissue looks terrible.
@@damienharkin last reply!! Haha, I got it, I needed a little more xylene in my old permount! Thank you Dr. Harkin!
@damien harkin is there any way you could list all the epuipment you used?
Hi Donna, equipment for performing H&E staining depends a little on how many slides and how often you intend to stain.
The simplest list of equipment (assuming that you already have sections on slides) would be:
1. Coplin jars, including at least two that are compatible with xylene (e.g. glass).
2. Xylene resistant forceps for handling slides.
3. A sink with overhanging staining rack (e.g. metal grid oven shelf).
4. A well ventilated area to perform coverslipping. Preferably a fume cupboard or down-draft bench, but if only occasionally performing a few slides, a fan may be sufficient.
5. Solvent waste containers.
In terms of key reagents.
1. Xylene
2. Absolute ethanol
3. Pre made Hematoxylin solution.
4. Hydrochloric acid
5. Ammonia
6. Deionised water
7. Pre made eosin solution
8. Plastic mounting medium
9. Glass coverslips
10. Paper labels
11. Graphite pencil for labelling slides prior to removing paraffin.
That should get you started. Happy to provide more details if required.
Sir I have a question about bone calcification: For unfixed bones, which of the following solutions should be used: formalin - nitric acid solution or formalin - formic acid solution? Please help me, thanks a lot.
Hi Linh,
Nitric acid is a stronger acid and so decalcifies bone more quickly, but it is also more damaging. So if starting with non-fixed bone, I would suggest combining the formalin with the weaker and therefore more gentle formic acid.
Thank you Sir for this wonderful video. I'm planning to do an H&E on liver tissue and I was wondering if I can have a written version of your protocole. Thank you
Hi Paule,
1. Remove paraffin from slides by immersion in two changes of fresh xylene for 3 minutes each.
2. Rehydrate slides by passing through 100%, 100%, 90% then 70% ethanol for 1 minute each, then rinse slides in tap water, followed by distilled water.
3. Apply your hematoxylin solution of choice.
The time depends upon the formulation used.
If using a regressive hematoxylin solution (e.g. Ehrlich or Harris) then you should apply for 10 minutes, rinse off in water, treat briefly with acidified alcohol (e.g., 3 dips in and out; see details in my videos), followed by dilute ammonia.
If using a progressive hematoxylin (e.g. Mayer's) then you need to apply for approximately 1-2 minutes, but check after each minute until desired level of staining is achieved. You may have to treat briefly with acidified alcohol (i.e., differentiate the slides) if they are too heavily stained. In all cases, once the desired level of hematoxylin has been achieved, treat with the dilute ammonia solution to stabilize binding of hematoxylin to the tissue and to convert it to a blue color.
4. Rinse slides in 90% alcohol.
5. Stain with eosin for 2 minutes.
6. Dehydrate slides by rinsing thoroughly but briefly in 90%, 100%, 100% ethanol, then place directly into xylene to clear for 1 min.
7. Transfer slides to second xylene container before coverslipping using a plastic mounting medium.
Hope that helps
Good luck!
@@damienharkin Thank you so much ! I’m working on vibratome section ( 40um) , do you think it could impact the staining and if yes, how could I adjust it ?
This is a very thick section! You will have trouble seeing details clearly by bright field microscopy. Standard thickness for H&E stained sections is generally between 2-4 µm. Do you have access to a microtome like that displayed in my other video?
@@damienharkin In my lab we just have a vibratome unfortunately. But I can ask to histology-microscopy platform. Can I have the link of the video where you talk about microtome ? Thanks
Introduction to microtomy video is here: th-cam.com/video/MRIQZPCX45I/w-d-xo.html
Hello, what are the most widely used fixative in HandE staining method
A. Formaldehyde
B.zenker formol
C. Picric acid
D. Osmic acid
Please help me answer
Hi Rica,
Neutal buffered formalin (3.7% w/v formaldehyde) should be fine for most H&E applications.
Thanks for watching.
D
Hi thank you for the quick answer, unfortunately my question lacks tatement it should have EXCEPT, everything is right except.
What are the most widely used fixative in H and E
" EXCEPT " PLEASE HELP all answer seems right
Oh, that's easy. You wouldn't use osmium. That's used for electron microscopy. Sounds like you are studying for an exam? Good luck!
Ohh God thank you so much ♥️♥️♥️ I am . Thank you so much it means so much
Thank you for a great video. How do you prepare the acid-alcohol and ammonium?
Prepare acid-alcohol by adding 5 mL of concentrated Hydrochloric Acid to 995 mL of 70% ethanol.
Prepare dilute ammonia solution by adding 1 mL ammonia to 999 mL deionised water.
@@damienharkin thank you Damien;you help me so much......
@@damienharkin I don't have ammonia...can I use sodium bicarbonate!
Yes. Any slightly alkaline solution should be fine.
Thank you. There are few videos for histology!
My pleasure. Am working on one for Gram stains at the moment.
@@damienharkin I am histotechnician student. We're still on processing and fixation. We'll be moving to embedding next week. Thank you. Gram stains? They work similarly to how gram positive and negative works right?
Yep, that's right. Gram stains use a complex of Crystal violet and Iodine to stain positive organisms. The negative organisms are generally stained using basic fuchsin or neutral red. It can be tricky since careful rinsing in an organic solvent is required to clear the negative organisms of Crystal violet/Iodine. So controls are essential.
@@damienharkin Doesn't that go in microbiology under clinical laboratory sciences? I tried going into medical lab as a technician (had some issues and personal stuff come up). I may do that after I am an HT as a bachelor's degree and become a laboratory generalist. That is interesting. I never knew how gram stains worked... only that some microorganism are listed as gram positive or negative, and that the gram negative ones tend to be very dangerous and hard to culture. Thank you so much for your replies! Are you a biologist? Cytologist? What field are you in and what is your job title? This is all so interesting and new to me!
I'm a professor at the Queensland University of Technology (QUT) in Brisbane, Australia. You can learn more about the courses that we teach @QUT via the links in my profile.
i would like to see the end result
thank you
You can view some outcomes of staining here:
histopics.tumblr.com/
thank you
what is the thickness of these sections?
3 µm