RE: [Histonet] prolonged impregnation in Golgi-Cox (Kolb) staining

From:"Lee & Peggy Wenk"

I don't know if the tissue can be "saved", but try washing in water for a
few hours then process as usual for this technique, and see what happens

A lot of things are working against you. The dichromate has been oxidized,
the mercuric chloride has released a lot of hydrochloric acid which has been
chewing up the proteins. And, boy, do you have a lot of fixative cross-links
from both the chomate and the mercuric that you normally wouldn't have.

As for the green color, I think that's due to the normally orangish/red
potassium dichromate Cr+6 being oxidized to green Cr+3 in an acidic
environment. The oxidizer is the air in the container, and the acidic
environment comes from hydrochloric acid being released from the mercuric
chloride during cross-linking with the tissue.

I have a couple of questions, for anyone in the Histonet community. This
Golgi-Cox fixation procedure comes up a on occasion on Histonet, mostly from
researchers. How do these labs dispose of the chemicals afterwards? Mercuric
chloride cannot be dumped down any sink, and most (but not all) water/sewer
treatment plants won't allow potassium dichromate to be disposed down the
sink either. And how do researchers dispose of the water and alcohol and
xylene in the processing afterwards, where mercuric chloride and potassium
dichromate are being pulled out of the tissue into the processing solutions?

Can't these tissues be fixed in formalin (or some other less toxic fixative
than mercury and chromium), and then IHC procedures done, such as antibodies
against GFAP or neurofilaments or NSE? I work in a hospital, and am just
wondering why this Golgi-Cox procedure is needed by researchers, but doesn't
seem to be needed by clinical histology laboratories.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
[] On Behalf Of Olek
Sent: Thursday, December 13, 2007 3:31 PM
To: Histonet
Subject: [Histonet] prolonged impregnation in Golgi-Cox (Kolb) staining

Dear Histonetters,

I have some brains which were left in Golgi-Cox chromation solution
(K2Cr2O7, KCrO3, HgCl2: about 1% of each) for over 6 months. I would really
like to use this material even if staining would not be very clear.  
Do you have an idea how to process the tissue in this case? Any help would
be appreciated (and I know, next time I will be more careful about where I
leave my preparates).
By the way, I have already cut one of these brains and I found the sections
to be dark green. I have noticed this colour in my G-C preparates before but
mainly on the surface. I am actually curious whether the tissue should be
more green or more red when I cut it. I mean should I shorten the normal
period of chromation (about 14-15 days) if I see the brain is getting green?

Yours truly
Olek Michalski
        Laboratory of Neurobiology
       of Development and Evolution

  Nencki Institute of Experimental Biology
  ul. Pasteura 3, 02-093 Warszawa,  Poland
  Tel. +48 22 5892268,  Fax +48 22 8225342

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