I am having trouble obtaining an acceptable Nissl stain of passerine brains
using cresyl violet. The birds were transcardially perfused, brains removed,
postfixed and cryoprotected before sectioning at 40 um on a sledge
microtome. The protocol I am using was originally used for rat brains and is
as follow:

    ddH20 (few dips)
    cresyl violet (5-10 min)
    ddH20 (rinse excess stain)
    70% etOH (few dips)
    95% etOH (few dips)
    95% etOH + acetic acid (few dips)
    ddH20 (few dips)
    95% etOH (few dips)
    100% etOH (few dips)
    repeat previous steps as necessary
    Xylene (one dip)
    coverslip

I am unsure as to the concentration of cresyl violet I am using and it has
sat in a fridge for >2 yrs (although I have been assured that the age should
not be a problem). I have tried playing with the protocol quite a bit,
varying time in cresyl violet or how many times I dehydrate and
differentiate. I am getting near my wits end and still cannot get a good
stain. All my sections look as if they are washed out and I often have some
stain 'smeared' under the cover slip after dipping in xylene.

Can anyone help me? Thanks in advance!

Brandon
_____
Brandon Munk
Department of Zoology and Physiology
University of Wyoming
PO Box 3166
Laramie, WY  82071
_____
bmunk@uwyo.edu


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