Re: [Histonet] Cresyl Violet stain for bird brains

From:Maria Mejia

Hello Brandon,
The following cresyl violet acetate stain is one I have used on several 
animal species . What I found is that the pH is very critical in making 
this stain.
If it's not right will result in NO staining!
CV stock solution: (I purchase my crysyl violet acetate dye from Fluka 
You can order this dye from Sigma 800-325-3010 or see website).
Dist  H20 - 75ml
Mix well in a brown bottle. I let it mix- overnight
Then make up the acetate buffer A & B solutions:
Sol A:
glacial acetic acid- 3ml
DH20 - 250ml
Mix well.
Sol B:
sodium acetate-0.6gm
DH20 - 75ml

Working Sol. of CV stain
Sol. A - 237ml
Sol. B - 63ml
Add CV stock from 50ml to 70ml (I sometimes use 50ml to 60ml)
Mix well in brown bottle and pH 5.0 - this is very important.
Filter before use once a day & check pH too!!! This dye solution is 
stable & can
keep at RT .

Staining w/CV:
I usually stain 1 or 2 slides for testing.
Defatt in xylene (to remove lipid membrane from cells) - 1 change 10 
dips - 20mins.
hydrate in 100% (ethyl or reagent) alcohol) - 3 changes  - 10 dips ea. - 
4 mins ea.
95% alcohol - 1 change 10 dips - 4mins.
70% alcohol - 1 change 10 dips - 4mins.
Cresyl violet acetate stain - 10 slow dips - stain 30 sec - 2 mins.
**remember fresh stain is a bit stronger.  Try a 1 minute staining time.
70% alcohol - 10 dips - 1min. (sometimes might need to do this step for 
(during the staining procedure this alcohol must be changed so the 
alcohol looks
clear and not blue. If blue color, it will stain your sections further).
95% alcohol - 10 dips - 1 min.
100% alcohol - 3 changes - 10 dips ea. + 4 mins ea.
**should see a nice clear difference between the grey & white matter.
xylene - 3 changes - 10 dips ea. + 5 mins ea.

I hope this helps.

Maria Bartola Mejia
Smith-Kettlewell Eye Res. Inst.
San Francisco, Ca


Brandon Munk wrote:

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