THE QUESTION OF TRIAZENES IN THE TOTAL COLOR EFFECT OF THE PROTEIN AZO COUPLING REACTION IN HISTOCHEMISTRY

The suggestion that triazenes formed by union of alkalized diazonium salts with aliphatic amino groups contribute to the azo color reaction of tissue proteins is rejected. Deaminations of formaldehyde-fixed and other tissues were sufficient to render the previously oxyphil elements of these tissues...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry 1970-03, Vol.18 (3), p.187-194
Main Author: LILLIE, R. D
Format: Article
Language:English
Subjects:
Animals
Azo Compounds
Diazonium Compounds
Ethylamines
Guinea Pigs
Humans
Hydrazines
Hydrogen-Ion Concentration
Hydroxylamines
Proline
Proteins
Rats
Staining and Labeling
Sulfates
Triazines
Uric Acid
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Summary:The suggestion that triazenes formed by union of alkalized diazonium salts with aliphatic amino groups contribute to the azo color reaction of tissue proteins is rejected. Deaminations of formaldehyde-fixed and other tissues were sufficient to render the previously oxyphil elements of these tissues basophilic to azure A-eosin B at the same pH level. After these deaminations there was no detectable difference in intensity or distribution of the azo coupling reaction. Freshly diazotized safranin O, dimethylphenosafranin (methylene violet) and the diazosulfanilic acid, azure A sequence technic were used as testing methods. Acid extraction even when prolonged to 24 hr and when acid concentration raised from 0.1 N to 0.24 N does not alter the intensity or distribution of the several azo coupling reactions as compared with unextracted preparations. Deliberate creation of triazenes by admixture of proline, diethylamine, hydroxylamine, hydrazine sulfate, glycine and uric acid, added to the diazo in stoichiometric excess, more or less completely inhibited azo coupling of all tissue elements. Uric acid and hydrazine were the most efiective, and then hydroxylamine, proline and diethylamine in that order. Reacidification of the uric acid compound to pH 2.2-2.5 for 10 min, filtration to remove liberated uric acid and realkalinization moderately restored the azo coupling capacity of diazosafranin. This illustrates acid destruction of a triazene of this diazotate. It is concluded that triazenes with tissue aliamino groups play no significant part in the final color effect of azo coupling with the diazonium salts used.
ISSN:0022-1554
1551-5044
DOI:10.1177/18.3.187