Problem with cresyl violet staining

From:"Knight, Brandon"

I'm having a problem staining fixed mouse brain w/ cresyl violet.  The
sections were 40um each.  Here is the protocol that I followed:

Mounted tissue on subbed slide (from PBS)
Dried for over a week
Rinsed slide in ddH20 for 2 seconds
Dehydration (4 minutes in each)
    70%EtOH + 95%EtOH(X2) + 100%EtOH(X2) + Zylene(X3) + 100%EtOH(X2) +
95%EtOH(X2) + 70%EtOH
Rinsed briefly in ddH20
Stained in 70C 0.1% cresyl violet (0.08% acetic acid) for ~1.5 min
Briefly went - ddH20,70%EtOH,95%EtOH,100%EtOH

Four different sectioned brains were stained at the same time.  After
following the above protocol, two were sufficiently stained and the other
two were not.  I tried to stain these two repeatedly, keeping them in cresyl
violet for up to 5 minutes.  The corpus callosum was deeply stained but the
rest would not stain at all.  What are the possible reasons for why this is
happening.  It seems like maybe the fat is not being dissolved sufficiently,
but then why did the other two stain properly.

Brandon Knight
Children's Research Institute
Center for Genetic Medicine
Children's National Medical Center
Washington, D.C.




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