USE OF THE DIAZOSULFANILIC ACID, pH 1 AZURE A SEQUENCE ON ADRENAL MEDULLA AND THE EFFECTS OF PRIOR OXIDATION AND REDUCTION ON THE REACTION IN SEVERAL SPECIES

With the diazosulfanilic acid, pH 1 azure A sequence adrenal medulla exhibits areas of pale green cells with green nuclei alternating with other areas grading from green to dark blue. Adrenal cortex and capsular collagen color greenish yellow, nuclei and elastin green, smooth muscle yellowish green and erythrocytes greenish brown to greenish black. The reaction is obtained best after 5% glutaraldehyde, then 10% formol then glyoxal, quite well with formol-free sublimate and lead acetate fixations and fails after Zenker and Kose bichromate formol fixations. Staining is prevented by prior oxidation (quinonization) with 10% iodine/methanol (1-2 hr 3°C), 0.1 M FeCl 3 (2 hr), 3-5% K 2 Cr 2 O 7 (2-3 hr) and 1% H 5 IO 6 (10 min) and after these oxidations is restored by 4 hr 5% dithionite (N 2 S 2 O 4 ), metabisulfite (Na 2 S 2 O 5 ) or thiosulfate (Na 2 S 2 O 3 · 5H 2 O) reductions. The reaction has been demonstrated in man (Homo sapiens), monkey (Macaca mulatta), hog (Sus scrofa dom.), dog (Canis familiaris), cat (Felis cattus), guinea pig (Cavia cobaya), rat (Rattus norvegicus albinus), mouse (Mus musculus albinus), gerbil (Meriones unguiculatus) and opossum (Didelphis virginiana). In the hog most of the adrenal medulla gave the dark blue reaction, fairly large areas with some segregation into islets of dark blue occurred in man, monkey, cat, dog and opossum and islets are seen in mouse, smaller in rat and inconstantly in the guinea pig and gerbil. It is thought that the reaction is showing noradrenaline. The method is not specific for this substance; certain proteins as hemoglobin and keratohyalin as well as enterochromaffin cells react strongly.