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

[John Fernandez]

John thanks again, I should have been more clear:

the method advocated by the FD Neurotech kit does involve freezing the
tissue, cutting (100-200um thick) sections on a cryostat and slide
mounting on gelatin-coated slides (this is all carried out 2 weeks post
incubation with the Potassium Dichromate + Mercuric Chloride/Potassium
Chromate solutions.

After the sections are slide mounted, they are left to dry in the dark at
room temeperature for 1-2 weeks after which they undergo the staining
procedure with solns C & D.

The kit then advises dehydration in an ethanol gradient followed by xylene
and coverslipping with a resinous mounting medium (we use DPX).

Can you suggest any intermediary step between immersion in solutions C+D
and dehydration/mounting that might halt the stain degradation that occurs
post cover-slipping?

Thanks in advance,

John




> 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 >>
>> >> --
>> >>
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>> >
>> --
>> John Fernandez
>> Research Assistant
>> Department of Medicine
>> University of Melbourne
>> Austin & Repatriation Medical Centre
>> Heidelberg, VIC 3084
>> Ph: (03) 9496 3257
>


-- 
John Fernandez
Research Assistant
Department of Medicine
University of Melbourne
Austin & Repatriation Medical Centre
Heidelberg, VIC 3084
Ph: (03) 9496 3257