[Histonet] Cresyl Violet stain for bird brains

From:Brandon Munk

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)

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 Munk
Department of Zoology and Physiology
University of Wyoming
PO Box 3166
Laramie, WY  82071

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