[Histonet] cresylechtviolett (cresyl fast violet) staining problems

Hernan Aldana Marcos haldana <@t> unimoron.edu.ar
Mon Dec 1 10:52:00 CST 2003

Dear Sandy, sometimes is not necessary (depends of the quality of the stain)
the adition of acetic acid to the alcohol, do the differentiation only with
pure 96% alcohol. Remember to make the deshidratation in ol 100 very
quiclky. The xilol no decolorate the section. Another form is: do te
deshidratation in the oven al 58°C and then put the slide directly in xilol.
I do it, if the alcohol decolorate very quickly my sections.

Sorry for my English....

This is our modification of Burk's stain (1969). This stain is used to show
Nissl bodies and cellular patterns.

1- Paraffin sections 15-20mm

2- Dewax sections in xylene, hydrate through graded ethanol to water.

3- Stain for 3 to 5 minutes in cresyl violet solution (See appendix).

4- Wash in tap or distilled water.

5- Differentiate in 96% ethanol. The differentiation must be checked under
microscope, to obtain only colored Nissl bodies and nuclei. If
differentiation becomes difficult,  sometimes it is necessary to add some
drops of 0.1% glacial acetic acid in 96% ethanol to accelerate this process.
If acidified ethanol is used, wash in several changes of distilled water and
then begin the dehydration by quick immersion in 96% ethanol.
Differentiation may be stopped in distilled water when several slides are
simultaneously being processed.

6- 100% ethanol (2 minutes), clear in xylene and mount in Eukitt.

Cresyl violet solution                              Cresyl violet

(Kresylviolett-Merck)                          0.1g.

                                                            Distilled water

                                                            Acetate buffer
0.2M pH 3.6                   50ml.

Dissolve the cresyl violet in the distilled water. Add the acetate buffer,
mix the reagents and filter before using. This solution is very stable and
may be used for 5 months with excellent results.

From:.Biocell 1996. 20(3): 265-272. Standardization of Fixation, Processing
and Staining Method for the Central Nervous System of Vertebrates.H J.

Dr. Hernán J. Aldana Marcos
Facultad de Medicina. Universidad de Morón
Machado 914. B1708JPD. Buenos Aires. Argentina
e-mail alternativo hernanjavier <@t> yahoo.com
web: http://hjaldanamarcos.bravepages.com

----- Original Message -----
From: "Sandy Thevarkunnel" <sthevar <@t> bu.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, December 01, 2003 12:33 PM
Subject: [Histonet] cresylechtviolett (cresyl fast violet) staining problems

> Hi,
> We just bought new cresyl fast violet from Cell point scientific to
> replace our old cresyl fast violet from Roboz which was produced in
> 1995.  The new stain seems to wash away from the tissue when we put it
> in the 95% ethanol with acetic acid step rapidly, but prior to the step
> there does not seem to be any detectable differentiation during our 75%
> ETOH, 75% ETOH with acetic acid and the 95% steps that precede the 95
> with acid step.  Our protocol for making the cresyl fast violet is to
> add 2.5g to 500mL deionized H2O stir and then filter.  This recipe was
> great with the old stain but the new does not seem to be working.  We
> are staining formalin fixed human brainstems cut at 50um.
> Staining protocol we use
> Defatting- 3hrs in 1:1 Chloroform:Ethanol
> Hydration: 7 minutes- 2x 100% ETOH, 2X 95%ETOH, 1x70%ETOH
>                      10 minutes- dH2O
> Staining- 4-6 minutes in CV
> 30 seconds dH2O
> Differentiation from 75%, 75% with Acetic acid, 95%, to 95% with acetic
> acid, variable times from 1 minute to 4 minutes
> Dehydration- 2x 100% ETOH for 4 minutes
> Clearing- 3 changes of Xylene 2min, 1min, 30 sec.
> Coverslip with Permount
> Sorry for the long message I was just hoping if it wasn't the recipe it
> just might be our protocol so if anyone has any ideas I'd really
> appreciate them, I'm kind of new at this.  By the way our old CV was
> black but the solution was still purple and the new CV is green and
> solution is the same purple colour but it seems thinner.
> Thank you very much,
> Sandy
> Boston University School of Medicine
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