Lorraine-

I feel that your dehydration/clearing sequence was designed for much thinner
sections than 40 u.  I realize that leaving your slides in EtOH longer will
tend to decolorize your staining some, but I would try doubling the times
you are using for these steps.   I would try staining longer, perhaps 5
minutes in the cresyl violet if too much staining is lost.  If the
decolorization is still too great, at least leave the slides longer and in
more steps of xylene.  With such a brief dehydration and such thick
sections, I think your xylenes will soon become saturated with water (5%
water in xylene will cloud it), and the xylene should not reduce your
staining at all.

Good luck!

Joe

Joseph A. Saby, BA, HT(ASCP)
Pfizer Global Research and Development
Drug Safety Evaluation
2800 Plymouth Road
Ann Arbor, MI 48105
Phone: (734)-622-3631
FAX:   (734)-622-3866
E-mail: joseph.saby@pfizer.com


-----Original Message-----
From: Lorraine Masse [mailto:lmasse@biosupport.com]
Sent: Monday, September 25, 2000 4:27 PM
To: 'histonet@pathology.swmed.edu'
Subject: FW: Cresyl Violet Staining



> 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