I'm using this protocol, but with charged slides:
http://www.aatbio.com/products/protocol/23062.pdf
The sections are from mice that were perfused with 4% PFA and post-fixed in 4% PFA overnight, cryoprotected in 30% sucrose, and cut at 20 um on a cryostat.
Anyone know why the sections are falling off during the potassium permanganate step?
Dear Jeremy,
It is always such a pain when sections detach! I am not sure with this protocol, but potassium permanganate is a pretty harsh chemical, so I can see where sections that are not well attached could detach. I have to say that charged slides are not always better than gel-coated slides in the attachment department, which might be why Surgipath/Leica has just released the Apex Superior Adhesive slides (I have no economic interest in these slides).
One researcher here on RG used poly-lysine coated slides with her paraffin sections that she stained with Fluoro-Jade, so perhaps poly-lysine might be an alternative to charged slides: http://www.ncbi.nlm.nih.gov/pubmed/20528171?dopt=Abstract
I looked at the 2005 Schmued paper, and he does make a point of using a particular gelatin to coat the slides (1% pig skin gelatin type A 300 Bloom) and baking the sections onto the slides after sectioning. He also uses free-floating sections that he mounts on with distilled water, and in my experience those sections are usually better attached than cryostat sections. Since he cuts his sections at 25 um, you might try using free-floating sections you cut in the cryostat, rinse well to remove the OCT compound, and then mount onto the slides. Dry them well (the original Fluoro-Jade paper let them dry overnight, I think), and see if that works.
If you don't want the hassle of free-floating sections, then you might want to try gle-coating some slides, since he does mention in the discussion that they have used cryostat sections.
Good Luck!
Jill
How did you dry your slides? RT over night or at 60°C? The protocol allows drying at higher temperature, which usually enhances holding of sections.
regarding charged slides: They have an expiration date, they have to be stored in closed boxes, one must not touch the surface, different brands have different qualities.
You can try to do the permanganat-step horizontally - means cover the sections with sufficient amount of permanganat on a staining bridge. Then turn the slides up and let the liquid run off, let stand for a few seconds before carefully rinsing with water. Working one slide after the other brings the chance to mount an escaping section again on the glass with a painting-brush.
Thank you both for your answers!
Jill, I realize in retrospect that gel-coated slides would have been better, I simply didn't think about it when mounting the sections as they are already stained with DyLight-594 tomato-lectin and I am using many of them for imaging right off the cryostat.
Gudrun, I have tried a 'horizontal' approach at it gives me the least amount of falling off if I am SUPER careful during the potassium permanganate step. This may be my only option since all the brains are already cut and mounted.
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