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

[John Fernandez]

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
>>
>> --------------------------------------------------------------------------------
>>
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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