[Histonet] Losing Sections While Dehydrating

[Amanda Madden]

Hello Everyone!

I have searched the archives because I am sure this is a problem that has
been encountered before, but I have been unsuccessful in finding the right
thread. I ran an IHC series dilution on a new primary antibody for NeuN. I
am using 35 um rat brain sections that were cut on a cryostat and stored in
Cryoprotectant until ICC began. After finishing the ICC protocol, I mounted
the slides that were floating in phosphate buffered saline onto slides that
I subbed two weeks ago (.5% gelatin subbing). I allowed the slides to dry
from Friday afternoon until Tuesday afternoon. I tried to dehydrate them for
coverslipping and when they went into the dH20 bath, some of the sections
began to fall off of the slides. This continued in the 50% Alcohol bath. All
tissue that made it through those first two baths in the series stayed on
throughout, but I did lose quite a few sections, especially those that had
the lowest concentration of primary antibody. My experience has been that
highly concentrated primaries make the tissue a bit sticky, but the best
staining generally comes from the least concentrated. Does anyone know what
the problem might be? I think it might be a subbing problem but I'm not
sure. Thanks in advance.

Amanda Madden
Research Assistant

P.S. The subbing protocol that I used only called for one "dip" into the
gelatin solution, and didn't call for any type of acid wash. Also, our
cryostat is a Leica CM3050S and I was wondering if any of your labs have
established a working range in terms of humidity. Our lab has been
experiencing very high humidity levels (up to almost 80%), so we were
wondering if anyone has established that cutting cannot be done unless the
humidity is under x%.