John -

Just out of curiosity, will Garvey's stain look (visually) like Bielschowsky's?  (Even though the
methodology is different?)

Thanks for your help,
Miriam

Miriam Schroeder
Research Associate
Berlex Biosciences

--- "J. A. Kiernan"  wrote:
> The technique you describe (quoted below, with a
> reference to Garvey, Bigelow & Carpenter 1999) closely 
> resembles an earlier one published by Winsome Garvey 
> et al 1987, in J. Histotechnol. 10:245-247. It bears 
> no resemblance whatever to any of the techniques
> introduced by Bielschowsky.
> 
> The "parent" method would probably be either that
> of EP Samuel 1953 J. Anat. 87:278-287 or that of
> A Peters 1955 Quart. J. Micr. Sci. 96:323-328. 
> These are "physical developer" techniques for
> staining axons. (A physical developer is one
> that contains silver ions and a reducing agent
> together with a stabilizing colloid - in this
> case, gum mastic - to slow down the otherwise
> almost instantaneous reaction between them.)
> 
> The distinguishing feature of Garvey's method is 
> the use of a high temperature to allow a short 
> time and low silver concentration in the first 
> stage of the procedure. Garvey et al's paper in
> J Histotechnol 22:37-41 (1999) provides many
> additional technical tips as well as some good 
> discussion of how and why the method works, 
> and 31 references.
> -- 
> -------------------------
> John A. Kiernan
> Department of Anatomy and Cell Biology
> The University of Western Ontario
> London,   Canada   N6A 5C1
>    kiernan@uwo.ca
>    http://publish.uwo.ca/~jkiernan/
> ________________________________________________
> "Featherstone, Annette" wrote:
> > 
> > Here is the Alternate Bielschowsky Stain:
> > 
> > 1. Deparaffinize to DH2O
> > 
> > 2. Place sections in 0.1% silver nitrate solution, containing citric acid
> > (working silver nitrate) in a 65 degree C water bath for seven minutes. (I
> > preheat this solution for about 5min, in a plastic coplin jar)
> > 
> > 3. Rinse in 2 changes of DH2O, remove slides one at a time.
> > 
> > 4. Rinse in 95% ethanol, then 2 changes of AbsoluteEthanol.
> > 
> > 5. Immerse slides in filtered gum mastic for about 5 min. (We keep this in a
> > coplin jar and top it off as needed)
> > 
> > 6. Drain well and put into developing solution in a 65 degree C water bath
> > for 12- 13 minutes. (I start to check it at around 10 minutes, I also
> > preheat this solution for about 5 min, usually when I put the slides in the
> > gum mastic.
> > 
> > 7.Rinse in DH2O
> > 
> > 8. Dehydrate clear and mount.
> > 
> > SOLUTIONS:
> > 
> > 1% Silver Nitrate
> >       Dissolve 1gm silver nitrate in 100 ml DH20. this solution last for
> > months in a brown bottle in the frig provided it is not left
> >       at room temp.
> > 
> > 0.1% Silver Nitate
> >       Take 5ml of the 1%silver nitrate, dilute with 45 ml DH2O. Prepare
> > fresh each time
> > 
> > 1% Citric Acid
> >       1gm citric acid in 100 ml DH2O
> > 
> > Working Silver Nitrate Solution
> >       Add 0.1 ml of 1% citric acid to 40 ml of the 0.1% silver nitrate.
> > 
> > Gum Mastic
> >      Dissolve 2.5 gm of gum mastic in 100ml absolute ethanol. Filter before
> > use. May be topped off and reused.
> > 
> > Hydroquinone
> >     Dissolve 0.5gm Hydroquinone in 30 ml DH2O
> > 
> > Developing Solution
> >     Combine 10 ml filtered gum mastic, 30 ml Hydroquinone, add 0.9ml of 1%
> > silver nitrate
> > 
> > We got this stain from the Journal of Histotechnology/Vol. 22, No. 1/ March
> > 1999 and we have used it ever since with absolutely beautiful results
> > 
> > We always use sterile water but I guess DH2O might be fine too. This saves
> > hundreds of dollars per year, maybe more.
> >  Here is the Alternate Bielschowsky Stain:
> > 
> > 1. Deparaffinize to DH2O
> > 
> > 2. Place sections in 0.1% silver nitrate solution, containing citric acid
> > (working silver nitrate) in a 65 degree C water bath for seven minutes. (I
> > preheat this solution for about 5min, in a plastic coplin jar)
> > 
> > 3. Rinse in 2 changes of DH2O, remove slides one at a time.
> > 
> > 4. Rinse in 95% ethanol, then 2 changes of AbsoluteEthanol.
> > 
> > 5. Immerse slides in filtered gum mastic for about 5 min. (We keep this in a
> > coplin jar and top it off as needed)
> > 
> > 6. Drain well and put into developing solution in a 65 degree C water bath
> > for 12- 13 minutes. (I start to check it at around 10 minutes, I also
> > preheat this solution for about 5 min, usually when I put the slides in the
> > gum mastic.
> > 
> > 7.Rinse in DH2O
> > 
> > 8. Dehydrate clear and mount.
> > 
> > SOLUTIONS:
> > 
> > 1% Silver Nitrate
> >       Dissolve 1gm silver nitrate in 100 ml DH20. this solution last for
> > months in a brown bottle in the frig provided it is not left
> >       at room temp.
> > 
> > 0.1% Silver Nitate
> >       Take 5ml of the 1%silver nitrate, dilute with 45 ml DH2O. Prepare
> > fresh each time
> > 
> > 1% Citric Acid
> >       1gm citric acid in 100 ml DH2O
> > 
> > Working Silver Nitrate Solution
> >       Add 0.1 ml of 1% citric acid to 40 ml of the 0.1% silver nitrate.
> > 
> > Gum Mastic
> >      Dissolve 2.5 gm of gum mastic in 100ml absolute ethanol. Filter before
> > use. May be topped off and reused.
> > 
> > Hydroquinone
> >     Dissolve 0.5gm Hydroquinone in 30 ml DH2O
> > 
> > Developing Solution
> >     Combine 10 ml filtered gum mastic, 30 ml Hydroquinone, add 0.9ml of 1%
> > silver nitrate
> > 
> > We got this stain from the Journal of Histotechnology/Vol. 22, No. 1/ March
> > 1999 and we have used it ever since with absolutely beautiful results
> > 
> > We always use sterile water but I guess DH2O might be fine too. This saves
> > hundreds of dollars per year, maybe more.
> > 
> > 
> > -----Original Message-----
> >>> From: J. A. Kiernan [mailto:jkiernan@uwo.ca]
> >>> Sent: Friday, February 14, 2003 14:24
> >>> To: Nymeyer, Heather
> >>> Cc: 'histonet@pathology.swmed.edu'
> >>> Subject: Re: Bielchowsky stain
> >> 
> > > "Nymeyer, Heather" wrote:
> > >  ... The method being used is a 20 % silver nitrate
> >>> > solution for 20 mins followed by a 15 min incubation
> >>> > (in the dark) of ammoniacal silver. ... A few drops of the reducer
> >>> > is added to the ammoniacal silver and checked microscopically
> >>> > until fibers are seen.  At the same time the fibers are being
> >>> > developed the excess background precipitate is forming.
> > >
> >>> This is what to expect if you add formaldehyde to an
> >>> ammoniacal silver solution; it's one way of making
> >>> mirrors. In all the variants I've encountered (and most
> >>> bear little resemblance to the methods published by
> >>> Max Bielschowsky from 1904-1910), the sections are put
> >>> in the reducing solution after the ammoniacal silver.
> >>> There may or mmay not be a distilled water rinse
> >>> between these steps, and this is a critical feature
> >>> affecting the result. A small amount of ammonia-silver
> >>> complex must be carried over, either as free liquid or
> >>> not washed out of the tissue.
> >> 
> > The principle of the method is that silver precipitation
> > occurs fastest in the axons because these already
> > contain small amounts of colloidal silver, formed
> > during the immersion in silver nitrate. Eventually
> > there will be silver precipitation everywhere, hence
> > the need for microscopic control of the reduction
> > step. The rate of reduction is affected by a variety
> > of factors such as the concentrations of silver and
> > ammonia and the pH. (These are explained by Nauta WJH
> > & Gygax PA (1951) Stain Technol. 26:5-11 in a paper
> > about their Bielschowsky-like method for degenerating
> > axons.)
> > 
> > If you're staining normal axons there are easier
> > silver methods than those based on Bielschowsky's.
> > 
> > --
> > -------------------------
> > John A. Kiernan
> > London,   Canada   N6A 5C1
> __________________________________________________
> 
=== message truncated ===


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