[Histonet] Cresyl Violet
d-heiferman <@t> northwestern.edu
Wed Jun 10 13:37:23 CDT 2009
We are trying to stain 35 and 5 micron paraffin embedded sections of mouse
brain (fixed in 4% paraformaldehyde) using cresyl violet to visualize cell
bodies and are having problems achieving a sufficient stain intensity (too
We are using cresyl violet acetate from Sigma and we have tried using two
different stain recipes:
2% cresyl violet acetate in a 0.4M acetate buffer solution at pH = 3.7-3.9.
Heated to dissolve then cooled and filtered before use.
0.1% cresyl violet acetate in a 0.1 M acetate buffer solution at pH = 3.6.
Stored in the fridge and filtered before use.
Between these two recipes, the 0.1% solution had a lot more precipitate that
was left behind during filtering as compared to the 2%, which had some but
less. This may have been due to the lack of heating, but there was no
heating in the 0.1% protocol.
We used a standard deparaffination and hydration technique to stain. We
tried multiple bath lengths in xylene from 5 minutes to 2 hours, then we put
the samples down an ethanol gradient from 95% to 70% to 50% to water. Then
we tried different lengths of time for staining, from 2 minutes to 45
minutes, but the stain almost completely washed out on the rehydration steps
up the ethanol gradient. Also 70% ethanol on the up gradient has an acetic
acid concentration of 1%.
Does anyone have any suggestions for improving our protocol? We’re unsure
if we need to change cresyl violet concentration, pH, dehydration procedure
or something else. Any suggests are greatly appreciated.
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