Nissl stain dilemma

From:Stephanie Moore <>

Hello all...

Sorry to bore you with this question but here goes:  I have to mount
"huge" frozen squirrel brain sections onto gel-subbed slides for
subsequent Nissl stain.  They are 50 micron sections so they take awhile
to dry.  I had great Nissl staining when directly thaw-mounting the
sections onto the slides, the slices stayed on throughout rehydration
and staining BUT I had these awful air bubbles which caused half-moon
tears in my sections.  I ran across a little technique written in a
mouse brain atlas (Franklin and Paxinos) where they got rid of their air
bubbles by putting a drop of PBS onto the slide and then putting the
slice onto it.  I tried this and it seemed to work wonderfully, I let
the sections dry for 48 hours at room temperature and the sections did
not stay on through rehydration.  I was rehydrating for 3 minutes each
in xylene(clearing step)-100-100-95-95-70-70-50-25% ethanols then water
for 1 minute then Nissl stain for 20-30 minutes. By the time I got to
the second 70% I was getting wrinkling and detachment of the edges, with
the whole slice detaching by the water step.  I need to get rid of the
air bubbles AND have the slices stay on through processing.

Can I use a drop of old subbing solution or gel-alcohol or something
instead of PBS...has anyone overcome this problem/how?  Also, side
question:  Can subbing solution be stored in the refrigerator and reused
for later subbing, or does it need to be made fresh?

Your vast knowledge and help are greatly appreciated...

Stephanie Moore
Lab Tech/Mgr
Brandeis University

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