Re: [Histonet] gallocyanine stain for Nissl

From:John Kiernan

The procedure in Bancroft & Gamble is OK. You can
shorten the staining time to 2 hours by doing it
in a 60C oven instead of at room temperature. (A
higher temperature may shorten the shelf-life of
the staining solution. Has anyone tested this?)

The correct spelling is gallocyanine (because the
dye is an amine). The CI number is 51030 (CI
Mordant blue 10).

The chemistry of the cationic chromium complex of
the dye and its binding to nucleic acids is well
understood - supported by various studies more
recent than the two cited in B & G. This is a
truly excellent staining method for anyone who's
not in a hurry.
Its big advantages over most other Nissl stains
(such as cresyl violet, toluidine blue and neutral
red) are that there is no overstaining and the
colour resists most subsequently applied chemicals
such as alcohol-water mixtures and acidic
solutions of anionic dyes. Simple basic dyes,
intelligently applied, can provide fast Nissl
staining for small batches of slides. With
chrome-gallocyanine there is no need for speed or
intelligence; that's why I really like the method,
even though the quality of the colour (hue?
saturation?) is a rather bland greyish-blue.

The only shortcoming of the gallocyanine-chrome
alum method is that the staining solution begins
to deteriorate after 1-2 weeks. Because maximum
coloration is confined to the stainable substances
(in the CNS this means nuclear DNA and ribosomal
RNA) it's possible to do mass-production staining
without having to worry about shelf-life. A 400 ml
batch of gallocyanine-chrome alum solution can be
used repeatedly to stain several racks of slides
for a few days. Throw the staining solution out
when you've finished the batch, because if you try
to use it again two weeks later it will be no
good. (Guess how I found that out.)

The intensity of cytoplasmic staining with
gallocyanine-chrome alum has been shown by
scanning microspectrophotometry to be proportional
to the concentration of what we now call rRNA.
Einarson's 1951 paper, which is cited in Bancroft
& Gamble, is a good read, and it shows that there
was molecular biology 13 years before anyone had
seen a ribosome. By the late 1950s it was known
that cells contained more RNA when they were
making more protein. Einarson's method, dating
from 1932, did its bit and is still useful.

John Kiernan
Anatomy, UWO
London, Canada.
Elizabeth Chlipala wrote:
> I need to perform a gallocyanin stain for nissel on mouse lumbar spinal
> cord.  I have a protocol from bancroft and it says to stain for 18-48
> hours.  Can anyone out there give me any pointers on this stain.  I also
> need to order the gallocyanin and and the only reagent I see is
> gallocyanine is that the same thing?
> thanks in advance
> Liz
> Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
> Manager
> Premier Laboratory, LLC
> P.O. Box 18592
> Boulder, Colorado 80308
> Office: (303) 735-5001
> Fax: (303) 735-3540

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