[Histonet] Re: Golgi stain question/Rapid Golgi Stain (FDNeurotech)

Mon May 15 10:59:58 CDT 2006

Your kit's literature declares itself (partly). It
is neither rapid nor Golgi, and certainly not the
classical rapid Golgi method! It is evidently a
"Golgi-Cox" method, perhaps based on Sholl's
modification or one of Kolb's much more recently
published variants. These are used for looking at
dendrites (branching patterns, spines).  The
secret items C & D in the kit will be for the
alkaline reduction step that deposits the black
stuff. In published methods this is usually a 1:20
dilution of ammonium hydroxide (.880 ammonia) in
water. A kit could generate an equivalent solution
by mixing two components (such as any ammonium
salt and any stronger base - KOH, NaOH, Na2CO3) in 
a prescribed volume of water. The calculation is
an elementary one. 

To answer your question about fixation:

The mercuric chloride/dichromate/chromate mixture
serves as the fixative in methods of this type. 

You say that the kit advises moving the blocks
into sucrose; presumably this is for cutting
frozen sections. But you also talk about
dehydrating in alcohol to "stop the reaction".
Reaction-stopping with alcohol isn't part of any
Golgi or Cox method. With any histological
procedure involving dichromate as a fixative or
other agent, thorough washing in water (hours;
slowly running, or plenty of changes) is necessary
to remove unbound dichromate before going into
alcohol, with which there is a chemical reaction
that leads to precipitation of Cr2O3. 

John Kiernan
Anatomy, UWO
London, Canada.
_____________________________________-
John Fernandez wrote:
> 
> John, thanks for your reply;
> 
> the kit contents are of the Cox'x variety.
> That is, solution A of the "FD Rapid GolgiStain Kit" contains Potassium
> Dichromate + Mercuric Chloride.
> 
> Solution A is added to solution B (Potassium Chromate) and the brain
> tissue is left to be impregnated with this mixture for two weeks after
> which it is transferred to a sucrose solution for 4 days before being cut
> and slide mounted and put through further staining with solutions D & E
> (contents of which the company has not disclosed).
> 
> With the Cox's method that you know of, is there a fixation step (other
> than dehydrating in ethanol gradient and xylene) that is undertaken to
> stop the reaction? 
> 
> As I have mentioned we do get excellent initial staining but the problem
> is that it continues on even after coverslipping.
> 
> Thanks,
> John F
> 
> > The first thing you need to find out is whst's in
> > the kit.
> >
> > Is the method the one traditionally known as the
> > rapid Golgi method? This involves fixation in an
> > osmium tetroxide-potassium dichromate mixture, for
> > 2 to 7 days (the time affects the outcome, but
> > unpredictably) followed by immersion in aqueous
> > silver nitrate for a few days, and preparing thick
> > celloidin or paraffin sections. There are plenty
> > of later variants, having in common a silver
> > chromate end product, but these should not be
> > called "the rapid Golgi method". The sections are
> > mounted in thick Canada balsam (the real stuff)
> > without coverslips. If a coverslip is applied, the
> > preparations fade with time. There are various
> > chemical tricks to prevent such fading.
> >
> > The other major member of this group of methods is
> > Cox's, with mercuric chloride and potassium
> > dichromate, but no silver. The dark deposit in
> > this case is thought to be a mixture of mercurous
> > oxide and colloidal mercury. These methods are
> > often called Golgi-Cox, even though Golgi didn't
> > invent or use them. Ideally Golgi-Cox preparations
> > should also be mounted without coverslips, but
> > they will keep for a few years in DPX with
> > coverslips.
> >
> > John Kiernan
> > Anatomy,  UWO
> > London,  Canada
> > ____________________
> > John Fernandez wrote:
> >>
> >> Hi all,
> >>
> >> our lab has recently purchased and used the Rapid Golgi Stain Kit from
> >> FD
> >> Neurotech, resulting in very impressive staining down to dendritic spine
> >> level, however as Julie Heinrich found, within 24 hours this staining
> >> becomes very granular with loss of distinct morphology that was
> >> otherwise
> >> seen previously. It progressively worsens over time.
> >>
> >> Has anyone successfully used the FD Neurotech Rapid Golgi kit? Or does
> >> anyone know of a mounting medium that can be used to adequately stop the
> >> reaction that is taking place? Any ideas on what could be causing this
> >> progressive deterioration of staining would be very much appreciated.
> >>
> >> Thanks in advance,
> >>
> >> John
> >>
> >> John Fernandez
> >> Research Assistant
> >> Department of Medicine
> >> University of Melbourne
> >> Austin & Repatriation Medical Centre
> >> Heidelberg, VIC 3084
> >> Ph: (03) 9496 3257
> >>
> >> Re: Golgi stain question
> >> From: "J. A. Kiernan" <jkiernan <@t> uwo.ca>
> >>
> >> --------------------------------------------------------------------------------
> >>
> >> On Fri, 30 Mar 2001, Julie Heinrich wrote:
> >>
> >> > I am terribly sorry to bother you- I have been looking through the
> >> > histonet web page and have found several helpful replies from you
> >> > about the Gibb & Kolb Golgi stain method.  I haven't managed to
> >> > figure out how to properly post a question there -
> >>
> >> You can't. It's a collection of old (archived) communications.
> >> To ask or answer questions you have to subscribe to the
> >> listserver. This is done by sending an email to
> >>  histonet <@t> pathology.swmed.edu  with the one word  subscribe
> >> in the Subject line. Nothing else. You'll then get an automatic
> >> reply from the listserver telling you all about it.
> >>
> >> > I'm attempting to use the method on avian tissue. I have obtained
> >> > some decent looking tissue so far (though the stain is a dark
> >> > tan/brown, rather than the preferable black that I had expected) yet
> >> > after time the stain turns very 'grainy'. Within a matter of just 24
> >> > hours, the dendrites/spines look like collections of dots rather than
> >> > complete structures, and it gets worse with time.
> >>
> >> > Do you have any idea why this might be happening? (I'm following
> >> > their protocol to the best of my knowledge, and use fresh solutions
> >> > each time I run tissue)
> >>
> >> A graduate student here called Tim Ho did great numbers of Kolb
> >> Golgis on rat brains a few years ago. He went to Kolb's lab in
> >> Lethbridge, Alta for guidance. His initial problem was that the
> >> unstained spaces between the black cells were pale green and
> >> a bit granular. If I remember rightly, the washing after the
> >> Golgi-Cox solution needed to be more thorough.
> >>
> >> Your problem is different, and may relate to the mounting medium.
> >> Traditionally the sections were mounted in thick Canada balsam
> >> without coverslips. A modern synthetic medium + coverslip can
> >> be followed by fading, but I haven't heard of this happening
> >> in 24 hours. 6 months, yes. Are you cutting vibratome sections
> >> of adequate thickness? I think you must be doing something wrong
> >> at or after the sectioning step, but don't know what. Sorry I
> >> can't be more helpful.
> >>
> >> ----------------------------------------
> >> John A. Kiernan
> >> Department of Anatomy & Cell Biology
> >> The University of Western Ontario
> >> London,  Canada   N6A 5C1
> >>    kiernan <@t> uwo.ca
> >>    http://publish.uwo.ca/~jkiernan
> >>
> >> --------------------------------------------------------------------------------
> >>
> >> << Previous Message | Next Message >>
> >> --
> >>
> >> _______________________________________________
> >> Histonet mailing list
> >> Histonet <@t> lists.utsouthwestern.edu
> >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >
> 
> --
> John Fernandez
> Research Assistant
> Department of Medicine
> University of Melbourne
> Austin & Repatriation Medical Centre
> Heidelberg, VIC 3084
> Ph: (03) 9496 3257