[Histonet] Cresyl Violet Counterstain of LFB

Lee & Peggy Wenk lpwenk <@t> sbcglobal.net
Fri Jun 5 05:04:47 CDT 2009

Some thoughts:

1. Are you using cresyl violet, or is it cresyl violet acetate, formerly
known as cresyl echt violet. It should be the cresyl violet acetate.

2. 5 minutes may not be long enough. We use 1 hour.

3. After differentiation, the only things that should be violet are the
nuclei of glial cells and neurons, and the nissl substance in the body of
some neurons. And if you are doing the LFB first, the myelin changes from
baby blue to medium blue. So there really won't be a lot of violet seen in
some parts of brain, where there aren't a lot of neuron bodies. Just check
the glial cell nuclei for positive staining.

The following is our CEV (CVA), which can be used alone, like what follows.
Or, skip step 1, and after you finish the LFB, go directly to step 2, and
complete as written.

0.5 g Cresyl echt violet (Cresyl Violet Acetate) (no CI number)
0.18 g Sodium acetate (CH3COONaC3H2O)
500.0 mL Distilled water
1.5 mL Acetic acid, concentrated (CH3COOH)(approximately)
Dissolve cresyl echt violet and sodium acetate in distilled water. Slowly
add acetic acid, drop by drop, to solution. Should have a pH of 3.5. If
solution pH is below 3.5, add more sodium acetate. If solution pH is above
3.5, add more acetic acid. Filter. Let stand overnight before using. Store
at room temperature. Stable for months. May be reused until weak.

PROCEDURE - Cresyl Echt Violet:
1.	Deparaffinize and hydrate slides through graded alcohol to distilled

2.	Place sections in cresyl echt violet solution	1 hour (can
overstain, since differentiating out)

3. 	Differentiate in two changes of 95% ethanol until nuclei and Nissl
granules remain violet and the background is nearly colorless. Check
differentiation with the microscope. 1 to 2 drops of acetic acid many be
added to the first alcohol to speed up differentiation.

4.	Dehydrate through absolute ethanol and clear in xylene.

5.	Coverslip with a synthetic mounting media. 

Lee & Peggy Wenk
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Patten,
Nicole (NIH/NIAAA) [F]
Sent: Thursday, June 04, 2009 9:55 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Cresyl Violet Counterstain of LFB

Hi Histonet... Quick question!!

I'm doing Luxol Fast Blue staining on frozen human brain sections (1% LFB in
95% EtOH with Hydroquinone/Sodium Sulfite Differentiator) and trying to
counterstain with Cresyl Violet. Following Cresyl Violet incubation (0.1%
for 5') my protocol calls for me to dehydrate the tissue in ethanol (95%x2,
100%x2) but this pulls out basically all of my CV stain! Does anyone have a
solution for this? 

Also, I have horrible frozen artifacts... is there anything I can do even
though the tissue has already been sectioned? What about during the
sectioning process?

Thanks in advance!

Nicole J. Patten
Post-Baccalaureate Fellow/IRTA
NIAAA/National Institutes of Health

Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu

More information about the Histonet mailing list