Dear All,
I am trying to do immunohistochemistry on mouse muscle samples, the problem that I confront with floating out slice in water after 40 min in Tris-EDTA buffer antigen retrieval. Sections were cut 4 μm thickness and cooked 1 hour, 60 ℃. I will be grateful by receiving any comment on solving this problem.
For paraffin sections is it important to get rid of the water between glass-slide and section after mounting. After mounting dry in vertical position. Sometimes one can see a little "bag" of water made by the paraffin. this hampers the binding of the tissue to the glass. Drying should not exceed one hour at 60°C. Studies showed, that antigens are influenced by dry heat, but it has the advantage of better adherence of the section. Over night drying at room-temp and then one hour in the oven would be my choice. This also helps to melt the paraffin for deparaffination.
My experience also showed, that thicker sections are more easily detached from the glass. 4µm is fine.
With our automatic IHC stainer we have no problems with high pH retrieval, but it may be helpful to choose a milder way of application. Instead of blubbering-boiling a little bit lower temp in a waterbath for a longer duration may be better. Or a steamer.
Adhesive slides are essential. Be careful with these slides. The binding sites can be "destroyed" by finger-prints and too long exposure to light and air.
It could be a few things but my first inclination is to ask whether you are mounting the tissue on positively charged slides.
Hi,
- do you use superfrost slides?
- 4 um is very thin, it's best to do 10 um if you can
- air dry 1H at RT your sllide after cutting
I have used Superfrost slides from Thermo Science Co.
@Vincent: as I read, If cutting Slice be thick, the probability of detaching will be more.
Do you mean before cooking, one hour air dry?
By experience, cutting under 10 um increase the probability to loose the section
Yes, air dry your section before heating.
When a use a freeze slide, I air dry it 10 min and heat it 10 min at 50°C before immunostaining
thanks, but it seems your answer regards to cryosection, I use paraffin section.
Did you try new vs opened box of Menzel (Thermo) Superfrost. There is also a difference between Superfrost and Superfrost Plus.
I had some floating from Superfrost but none from Superfrost Plus, so far.
Also, I dry the sections, after cutting, for 2h at 55 C and then overnight at 40 C
No issues so far. Check these options too :)
Good luck!
Hi, Paraffin sections within 4-7 micron meter thickness will not drop, if you use positively charged slides. Hope you are not using Tris-EDTA for antigen retrieval? Muscle samples have any specific way to antigen retrieval, I am not sure. I have been using nervous tissues for last 20 years, I never come across this issue. To me, you sections may not attach properly after mounting it on the slides...let it dry for over night at room temperature. For the fun of it, after O/N dry, just do one slides with H&E staining to make sure, your sections attached properly.
For paraffin sections is it important to get rid of the water between glass-slide and section after mounting. After mounting dry in vertical position. Sometimes one can see a little "bag" of water made by the paraffin. this hampers the binding of the tissue to the glass. Drying should not exceed one hour at 60°C. Studies showed, that antigens are influenced by dry heat, but it has the advantage of better adherence of the section. Over night drying at room-temp and then one hour in the oven would be my choice. This also helps to melt the paraffin for deparaffination.
My experience also showed, that thicker sections are more easily detached from the glass. 4µm is fine.
With our automatic IHC stainer we have no problems with high pH retrieval, but it may be helpful to choose a milder way of application. Instead of blubbering-boiling a little bit lower temp in a waterbath for a longer duration may be better. Or a steamer.
Adhesive slides are essential. Be careful with these slides. The binding sites can be "destroyed" by finger-prints and too long exposure to light and air.
Just addition to previous comments, back in US times , i used paraffin embedded quail embryos (4-5ss) for sectioning. We used gelatin coated slides. Nowadays for skin samples, we use HistoBond slides.
I would recommend gelatin coated slides if you are having trouble with sections remaining attached. Commercially coated slides are pretty good, but coating them yourself so that the coating is fresh is even better.
I agree with Karen, make sure you're using adhesive slides -or ones you've coated yourself. I'm currently using Superfrost Plus slides and have no dramas even when doing Ag Retrieval at 105oC. Good luck!
Hello!
I agree with Stephanie regard Superfrost Plus, that are a good products.
Therefore the antigen retrival with EDTA often detachs the sections, it's a strong method, maybe you can try change buffer.
It is nice to inform, I tried by two different buffer citrate buffer and citrate-EDTA buffer, pH 6 and incubation time 30 min, and the problem was solved.
Thank you All
I would recommend Star frost slides (knittel glass) if you are having trouble with sections remaining attached. I get good adhesion to the slide. I also recommend pre IHC make a fixation with methanol / acetic acid 95% / 5% for 1-2 minutes at -20ºC; and I used the Antigen retrieval buffer citrate buffer and citrate-EDTA buffer, pH 6 and incubation time 4 min in pressure cooker.
I also think if your sample is OCT frozen then you need to fix your sections in cold Acetone for 7 min. Then there is noway that your sections washed away. I even keep my slides after fixation under running water for 5 min.
@Kamnoosh Shahabi. Recently I am trying to hybridize (FISH) my sample (the main material is Polyvinyl alcohol), which is cut in OCT frozen. After drying my sections at 46 degree, I apply probe and keep my samples in a moist chamber with hybridization buffer over night. I have the same problem that my samples detach from the slide. What kind of slide glass do you use? And could you explain why fix in cold acetone can solve this problem? Thank you
I have a similar problem with 20µm cryosection slices of mouse cornea: They wash away when incubating in 0.1M phosphate buffer (with or withour 0.5% Triton-X).
I use microscopic slides that I coated with gelatine myself. Strangely enough, 20µm slices of mouse eyelids (which are on the very same microscopic slide as the cornea slices) do not wash away... only the corneal samples do?
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