Re: [Histonet] Leder's Method (re: Sodium barbiturate)

From:"Bryan Hewlett"

Hi Doug,

We always obtained our Na barbiturate directly from hospital pharmacy (they
have a license!).
However, you have at least two options.
My first choice is;
1) Use The Napthol AS-D chloroacetate method of  Li, C. Y., Lam, K.W. and
Yam, , L.T. (1973).
"Esterases in human leucocytes". J. Histochem. Cytochem., 21; 1-12

Briefly, it is as follows;
Solution A
4% Pararosaniline in 2N HCl.................0.1mL
4% Aq. Sodium nitrite...........................0.1mL
Mix and add to Sorensen's phosphate buffer, pH 6.3......35.0mL

Solution B
dissolve 20mg Napthol AS-D chloroacetate in 1.0 mL N,N-dimethylformamide.

Mix solutions A & B, shake and filter directly into a Coplin jar.

a) sections to water
b) Incubate in substrate at R.T. for 2 hours (Mast cells are faster to come
c) rinse in water
d) counterstain in Mayer's hemalum for 2 minutes.
e) blue in Scott's 2 minutes.
f) wash, DCM


Or you can;
2) Switch to Burstone's Napthol AS-D acetate/Fast blue RR technique, this
uses 0.2M Tris buffer at about pH 6.8 (Must be below pH7.1).
Problem is that you have to aqueous mount in glycerin jelly!!!!!

Cheers my old China.


----- Original Message -----
From: "Doug Geddes" 
Sent: Thursday, February 26, 2004 11:10 AM
Subject: [Histonet] Leder's Method (re: Sodium barbiturate)

> Question for my technical specialist.
> Leder's method for neutrophilic myeloid and tissue mast cells for
> paraffin sections and blood films - Michaelis veronal acetate buffer
> requires sodium barbiturate.
> Sodium barbiturate can't be obtained in Canada or brought across the
> boarder.
> Any substitutions? Variations? Please help!
> Doug Geddes BSc. MLT
> London Health Sciences Centre
> London, Ontario, Canada
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