Re: FW: Cresyl Violet Staining

From:"L. Gibbs" <>

Hi Cresyl stainers-

Here is my "guaranteed" Cresyl method for frozen brain sectioned
at 40-100um:

Cresyl staining solution:
I use Cresyl from Chroma-Gesellschaft (via Roboz).
5 grams Cresyl
1 liter DH2O
0.2 grams sodium acetate
pH to 3.5 with acetic acid, filter

Following sectioning on the sledge, I mount thick brain sections
from Tris buffer (no saline). I avoid mounting from phosphate
buffers. The slides are "double-subbed" gelatin slides. After
drying horizontally for several minutes the slides are tilted and
dried vertically in a rack until they appear dry. When sections
appear dry, bake in an oven at 50C for a minimum of one hour.

I use glass jar staining dishes for this procedure because I am
using large 2x3 slides. Even with regular slides, I think the
staining is more uniform with the horizontal racks.
Begin staining in:
 100% EtOH 2 changes, 30 minutes each.
Defat sections in Xylene, 2 changes, 30 minutes each.
Hydrate sections through 100% EtOHx2, 95% x2, 80%x1, 70% x1, 2
minutes each. 
Several rinses of DH2O
Cresyl stain , 3 minutes
Differentiate sections in *70% EtOH for apx. 2 minutes (check
under scope).
95% EtOH, 2 changes, total 5 minutes.
100% EtOH 2 changes, total 5 minutes.
3 changes of Xylene, total 15 minutes. May use Xylene substitute
for the clearing and coverslipping.

* Some methods call for differentiation in Acetate buffer, pH

Good luck!

Lorraine Gibbs
Physiology & Biophysics

On Mon, 25 Sep 2000, Lorraine Masse wrote:

> > I am using Cresyl Violet Acetate to stain serial sections of macaque
> > brain.  This tissue has been fixed in formalin for over 6 months, frozen
> > sectioned at 40 microns, and placed in PBS with 0.05% Sodium Azide before
> > mounting on slides.  I have been running into problems with fat
> > displacement when I mount the slides onto coverslips.  The fat tends to
> > spread all over the slide and causes problems when viewing the slides
> > under the microscope.  The protocol for staining calls for 2 minutes of
> > hydration in deionized water, 1.5 minutes in the Cresyl Violet Stain, 4
> > rinses in tap water for 30 seconds each, run through alcohol at 80, 95,
> > 100, and 100 percent for 30 seconds each, 2 minutes in xylene twice, and
> > coverslip.  I have tried hydrating the tissue in alcohol at 100, 95, 70,
> > 50, and 35 percent before starting the deionized water step,  which caused
> > the stain to be too dark for counting neurons.  I have also tried fixing
> > the tissue in 40% formalin before starting the deionized water step, which
> > discolors the stain.  Do you have any other ideas that might be
> > successful?
> > 
> > Thank you,
> > Sarah McGrath

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