Abstract
The azo-coupling of enterochromaffin with some 30 diazomium salts has been explored, in regard to rate, concentration and pH. A technic of enterochromaffin cell counting in consecutive serial sections was employed and the counts related to those obtained in immediately adjacent serial sections reacted with the ferric ferri-cyanide reagent of Golodetz and Unna (6) as modified by Lillic and Burtner (8). This procedure enabled the equation of a considerable series of experiments with different diazotates on a succession of individual animals.
Structure of the diazonium salt proved to have a considerable influence on the azo coupling rate. Of the nitroanilines, nitroanisidines and nitrotoluidines, those with the nitro group para to the diazonium radical coupled rapidly, those with ortho nitro groups slowly. Also p-anisidine was more rapid than the ortho isomer.
In the specific cases of p-nitroaniline and of di-o-anisidinse fresh diazotates were found to be more rapid and effective couplers than commercial stabilized salts in similar concentrations. It is probable that other comparisons of the same sort would give similar results.
While azo coupling increased in rapidity with higher diazo concentrations, increasing density of background reaction decreased the demonstrability of enterochromaffin, so that optimal concentrations were often 1-10 mM, but lower with "rapid" couplers or higher with slow.
The second diazonium group of fast blue B is probably but little utilized in azo coupling with enterochromaffin under histochemical conditions, since di-o-anisidine treated with one molar equivalent of nitrous acid appeared almost if not quite as effective as when two molar equivalents were used.
Fast blue B appears to fall into an intermediate group of diazotates, being neither as fast as the p-nitro derivatives nor as slow as fast blue RR, BB or VB, for example.