We've been having very mixed and somewhat unpredictable results during some recent ICCs. We are float mounting immunolabeled zebra finch brain sections (40µ) and some scrub jay brain sections (also 40µ). In some cases, after only an overnight drying period after float mounting on gel-subbed slides, we are getting the edges curling up. This leads to cracking and at the very least folding when we dehydrate and coverslip. In some cases we are losing important experimental tissue.
I initially had subbed slides using a lab recipe that called for fairly low concentration of gelatin (under 1%?). I thought I had fixed the issue by subbing (and resubbing) slides at a much higher gel concentration. However, this does not seem to have worked in the latest round, though we seemed to have some success recently. (I apologize, I don't have the recipes in front of me at the moment, I may be off a bit on those numbers).
One thought I had was that we are mounting in PBS. Could the salts make the tissue curl? I discount this because I have mounted in PBS before without this issue.
Any suggestions?
If you are mounting in PBS, even at a low concentration it could be a dehydration issue with the salts as you suggest. Right now, I am mounting in distilled water with a small amount of 1XPBS (10:1), this is how I have always done it and it has always worked fine. You should not use slides that have been resubbed, the gelatin coating may not be uniform for the slides resubbed and will definitely be different from other slides subbed once. If you are sectioning with a microtome, I would also recommend gelatin embedding the brains prior to sectioning. This prevents the tissue from curling and really helps hold everything together, especially for coronal sections. I agree with Roxana, our lab uses delicate task wipes to remove excess mounting solution.
Thanks both for your answers.
One thing I should be clear about is that the tissue is laying flat when we are done mounting, but then is lifting off the subbed slide at the edges as it dried, prior to our dehydration step.
It does sound as if the PBS may be part of the issue here, and I'll be switching to ddH2O to see if that helps. Immersing the tissue in H2O seems to have helped on some of the sections that were curling.
I'd appreciate any other feedback from others, as well. Thank you.
Hello, I have been mounting my brain sections (50um) with a solution made with 2/3 of DI water and 1/3 of 1x PBS, and I have never faced any problem during the drying process, my sections are really attached to the super frost slides, and they can go through high temperatures during the antigen retrieval process with out falling off from the slide. I hope this may help you.
Also, check the pH of the solution you are mounting in.
Probably should be about 7.4, I've has this problem when it was off.
I think we were having an issue with overly concentrated PBS causing poor attachment to the gel-subbed slides. We seem to have fixed the issue.
Jesse, 50% 0.1M PBS + 50% diH20,,,,,add 3-4 drops of Triton X to 500ml solution. It will stop the curling and relax the tissue for mounting
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