[Histonet] Cresyl Violet

[Daniel Heiferman]

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
light).

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.

Thank you