[Histonet] Red chromogens [alkaline phosphatase labels]

[John Kiernan]

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
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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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