[Histonet] Re: Super/Frost Plus slides & wrinkled brain sections

[David A. Wright]

Hello All

I suppose it would be pointless to enjoin those who use
Super/Frost Plus slides to actually read the Instructions
included with every packet (I too get mine from Fisher
Scientific)?

Miles Cunningham (via Charles Scouten) remarks that
"Superfrost isn't good enough for adherence for free-floating
sections at ~30 microns".  Not "good enough" is perhaps more
suited to the procedure used by Neil Fournier - someone has
told him to mount from 0.1M PB.  I routinely mount 40um thick
frozen-cut & floating-processed rat brain sections to
Super/Frost Plus slides without any problems. Guess what?  I
follow the instructions.  

I quote: "Float... sections on... distilled water.  DO NOT add
 glue or subbing solution...  You may substitute tap water for
distilled... but if you begin to use tissue sections, use
distilled water."

Miles did note that adherence of thaw-mounted sections is
better than after immersion in buffer. But why the need for
buffer any way?  Salts in any buffer will compete
preferentially for charged sites on the treated glass
(likewise after APES treatment) over proteins in the section,
so that the latter cannot bond well.  

After, say, a floating DAB reaction, I dip each section in a
petri dish of 'house' deionized water for a few seconds,
rinsing out the brush at the same time, and mount to
Super/Frost in this water.  (Brain sections should be rinsed
individually and not left too long or differential (osmotic?)
swelling of various regions can add to the wrinkling problem.)
 I DO wick away excess water (with Whatman 3MM paper) in order
to prevent overlap of e.g. the hippocampus or ventricular
margins, since I'm interested in these areas, and find that,
if anything, it improves bonding to have as little water and
ion traces as possible.

I guess the other question is whether the "wrinkling" Neil is
really detachment.  If it is a shrinkage problem from
dehydrating, and he is not doing a counterstain (e.g. Nissl),
I suggest that he takes his thoroughly air-dried slides and go
directly to a water tolerant clearing agent such as CitriSolv
& then resin.  For most neurobiological studies, subcellular
detail is less important than the distribution & number of
different cell types.  I find this perfectly acceptable for
the markers he describes - NeuN , Brdu etc.

All the best
-David A. Wright, PhD
Section of Neurosurgery
University of Chicago