Fuchsine (Was: [Histonet] Red chromogens [alkaline phosphatase labels])

Fri Oct 1 14:20:32 CDT 2004

Point taken! I notice that Chroma do use
the correct English spelling of fuchsine.
Like nearly everyone else they spell
phloxin with a terminal e, which is wrong
because it's in the same group as eosin.
Chroma also put a wrong terminal e on their
English spelling of erythrosin; that one is
unusual.
              --- John Kiernan
------------------------------------
Gudrun Lang wrote:
> 
> Dear John,
> I think the German spelling is Fuchsin (without e). Please look at
> www.chroma.de at their catalogue.
> Your vixen is German "Füchsin" (fuechsin).
> 
> greetings from Austria
> Gudrun
> 
> ----- Original Message -----
> From: "John Kiernan" <jkiernan <@t> uwo.ca>
> To: "Rena" <RFail <@t> Charleston.net>; <histonet <@t> lists.utsouthwestern.edu>
> Sent: Thursday, September 30, 2004 5:29 AM
> Subject: Re: [Histonet] Red chromogens [alkaline phosphatase labels]
> 
> Dear Rena Fail,
> 
> Red alkaline phosphatase product.
> 
> Methods giving permanently mountable red products have been available
> for many years.
> Davis & Ornstein (1959) introduced hexazotized pararosaniline as a
> trifunctional trapping agent for naphthols released by hydrolysis of
> naphthol phosphates. Pararosaniline (CI 42500; CI Basic red 9) is one of
> the four components of basic fuchsine and is commercially available in
> fairly pure form. New fuchsine (CI 42520; CI Basic violet 2), which is
> closely similar to pararosaniline and also available in pure form
> (Horobin, 2002); It is currently preferred for making the hexazotized
> reagent.
> 
> Kits are sold, but the substrate solution is easily made in the lab. In
> a simple procedure published at the IHCWorld.com (2004) web site (see
> below for its URL), the working mixture is prepared from four stock
> solutions, which are stored at 4 C. Solution D is warmed to room
> temperature before using. The amount of levamisole (inhibitor of
> endogenous tissue alkaline phosphatase) is clearly stated.
> 
> The working solution is made immediately before using. Mix 10 drops each
> of A and B, then add 10 drops of solution C followed by 10 Kits are
> sold, but the substrate solution is easily made in the lab. In a simple
> procedure published at the IHCWorld.com (2004) web site, the working
> mixture is prepared from four stock solutions, which are stored at 4 C.
> Solution D is warmed to room temperature before using. I've annotated
> the instructions from IHCworld.com to simplify the weighing and
> measuring.
> 
> A.  New fuchsine (CI 42520), 0.2% in 2M HCl. (Conc. HCl is 10 or 12M)
> B.  Sodium nitrite (NaNO2), 0.4% and levamisole,(= [-]-tetramisole
> hydrochloride) 0.48% in water.
> C.  Naphthol AS-BI phosphate, 1% in 100% dimethylformamide.
> D.  0.05 M Tris-HCl buffer, pH 8.7.
> 
> The working solution is made immediately before using. Mix 10 drops each
> of A and B, then add 10 drops of solution C followed by 10ml of the
> buffer (D). A drop is assumed to be 0.05 ml.
> 
> Sections are incubated in the working solution for 10-20 minutes at room
> temperature. They are then washed counterstained as desired, dehydrated,
> cleared and coverslipped.
> 
> Lojda et al (1979, p. 74-75) warn that the working solution may be red
> or brown from colored impurities in new fuchsine or pararosaniline. Such
> solutions cause excessive yellow background staining. The yellow/brown
> impurities are the same ones that can make Schiff's reagent yellow
> (removable from Schiff with activated charcoal). Pararosaniline
> certified by the Biological Stain Commission is suitable for making
> alkaline phosphatase substrate mixtures because it is required to be
> substantially free of brown and yellow materials (Penney et al., 2002).
> 
> Probably any batch of basic fuchsine certified by the Biological Stain
> Commission will be OK for making the IHCWorld.com alkaline phosphatase
> substrate solution, because each of the four possible components of
> basic fuchsine has three diazotizable amino groups and their molecular
> weights are all quite close. I don't know of any published study that
> establishes this, so you're probably safest to go with either certified
> pararosaniline or with new fuchsine from a reputable dealer.
> 
> References.  (Please check them out if you can. The 2nd is easy enough!)
> 
> Davis & Ornstein (1959) J. Histochem. Cytochem. 7:297-298.
> 
> IHCWorld.com (2004) New fuchsin alkaline phosphatase substrate solution.
> http://www.ihcworld.com/_protocols/chromogen_substrates/AP_new_fuchsin_red.htm
> 
> Horobin (2002) Chapter 14 in Conn's Biological Stains, 10th ed.
> Oxford:BIOS.
> 
> Lojda et al (1979) Enzyme Histochemistry. Berlin: Springer.
> 
> Penney et al. (2002) Biotech. Histochem. 77:237-275.
> 
> Finally, please note that the correct spelling is fuchsine, not fuchsin,
> despite anything you might read in a catalog or on a bottle label. This
> is not a matter of American, British and German spellings. (Fuchsin is
> German for vixen; the dyes are, I think, named from their colours being
> similar to those of Fuchsia flowers rather than foxes.) Look in
> Websters, the Oxford, etc. It's fuchsine with an e. An -ine ending of an
> informal name of a chemical indicates that it is a base, often an amine.
> The -in ending occurs with compounds that are not bases, such as
> dextrin, eosin etc.
> 
>                   John Kiernan
>                   London, Canada
> ------------------------------------------------------------------
> Rena wrote:
> >
> > I have been having some trouble with some Abs stained with New Fuchsin
> > and permanent red. Recently I was asked if I were using Levamisole in
> > the permanent red and if so how much? I was told too much Levamisole in
> > permanent red would result in no staining. I have used 1 drop per ml for
> > both New Fuchsin and permanent red, which is the appropriate amount.  We
> > have run both DABS and either permanent  red or New Fuchsin with the
> > same AB from the same vial with vastly different results. Any ideas?
> > Rena Fail
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-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________