Hi all,
I've processed my own IHC for many years and never run into this problem before, but the last few times I've tried staining, I've had the same issue. I'm wondering if it's a reagent problem or something I'm doing.
We have macaque brain tissue that was fixed, cryoprotected, frozen, and sectioned at 50 microns. The sections were stored in PBS with azide before staining, all free-floating. After staining, I mounted them and let them air dry. They've been on the bench for at least a week while I waited for an afternoon to coverslip them. I'm using a standard protocol for dehydration of ascending ethanol concentrations (50%, 70%, 95%, 100%, 100%). On the first step, the 50% ethanol, I noticed a large number of bubbles forming under the tissue, between the tissue and slide. Gently pressing on them with a paintbrush can sometimes remove them, but obviously this is damaging the tissue and I'm worried that either removing the bubbles or leaving them as they are will result in poor tissue quality. I subbed the slides myself with gelatin, so my current suspects are the slides/gelatin, the alcohol, or the sections themselves. Anyone experience this before or know what might be causing it?
Hello Catherine,
there could be a lot of reasons which lead to such effects which you have discribed. My recommadations:
Do not mount the sections out of PBS because the phosphate salt is hard to solve after air drying. These salt crystals bind the water. And after cleatring in Xylene you can see these "air bubblers" which are actually non solved water bubbles.
Mount your sections out of 0.45 % NaCl plus a drop of dish washer solution (it's called Pril in Germany) if you use a normal petri dish for mounting.
Let the mounted sections air dry over night. Dehydrate them after a short rinse in distilled water (in this step you remove the salt crystals) and start with the dehydration in 70%, 2 x 100% ETOH, Isopropanol, 2 x Xylene, each bath 3 -5 min. (30µm thick sections 3 min is fine; thicker sections 50µm you can prolong the time up to 10 min).
If you like to change your gelatine protocol, here is the one which I have used since years:
Solve 0,6 g Gelatine and 0.06 g of KCrf(SO4)2 x12H2O (Chrom(III)-potassiumsulfate-Dodecahydrate) in 120 ml boiling distilled water.
Filter the solution, dip the clean slides, dry them in the oven at 37°C over night, put them in dust free boxes until use.
Good luck!
- Badges
- Science topic
- Similar topics
- Filing