Re: Paraffin processing brain slices

From:Lorraine Gibbs

Hi Bert-

We do a fair number of 300-500um brain slices (which have been used in
physiology experiments).
For best results, the slices must be fixed while weighted with either a
coverslip or smooth filter paper. We put the slices in the small (40mm)
petri dishes and place a round coverslip or smooth filter paper on top
before replacing the petri dish cover. Slices are fixed in either 4%PFA or
10% NBF for 6 hours-overnight at 4C.

The best way to section the slices is to do frozen sections with a sledge
microtome. If doing frozens, transfer the slices to 25% sucrose in PO4
buffer for 6 hours-overnight at 4C. Slices can be held in this solution for
up to a month prior to sectioning. To section on a sledge, make certain that
the platform is level. Use a carpenter's level. When the sledge platform is
frozen, pipette a small amount of the sucrose solution onto the platform in
order to make a platform for the slice. Take a few slices of the sucrose
platform with the knife before "painting"  the brain slice on to the
sucrose. The slice is now ready to section at 30um-100um.
The slices can also be sectioned on a cryostat that has a brain orientation

If you are stuck with paraffin, follow the fixing protocol, then process the
slices sandwiched into a cassette with embedding sponges. You can probably
shorten the processing times to 30 minutes. I hand-process without
additional heat or vacuum. I would use Histoclear or something similar and
avoid xylene. I have only used paraffin once-it worked but the slices do
look their best as frozen sections, preferably on the sledge if you have
access to one.

Good luck!

Lorraine Gibbs
Physiology & Biophysics
University of Washington
----- Original Message -----
From: "Bert Dotson" 
To: "Histonet (E-mail)" 
Sent: Wednesday, January 23, 2002 12:21 PM
Subject: Paraffin processing brain slices

> A pharmacology researcher here assures me that a number of published
> studies indicate paraffin processing of 300 micron thick (thin if you ask
> me) slices of rat or mouse brain. Of course the procedure reported is
> "slices are paraffin processed and sectioned." Has anyone out there done
> this? Any particular tips or tricks? I am assuming a support medium like
> agar is used prior to processing.
> Thanx in advance
> Bert Dotson

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