170 Microscopic Histochemistry 



the phosphates and sulfates are quite soluble in water; esters 

 of organic acids are very poorly soluble and require the ad- 

 dition of some acetone or propylene glycol to the incubating 

 mixture to obtain clear solutions. Fortunately, the substrate 

 concentrations required are very low ( 3-10 mg. of crystalline 

 substrate to a Coplin jar, or 0.00015-0.0005 M). 



Diazonium salts, which formerly had to be synthesized 

 freshly for each experiment, are now available commercially 

 in a wide variety and in a stabilized form. As dry powders, 

 they can be kept in the icebox, if protected from light and 

 moisture, almost indefinitely. However, their solutions are 

 quite unstable and should be used immediately. Some of 

 them keep better than others, but, on standing, even the most 

 stable will decompose within about 2 hours. The rate of de- 

 composition increases steeply with temperature and pH. The 

 decomposition products are dark and have a tendency to 

 stain the background, especially acidophilic structures, in a 

 murky brownish shade. If incubation must be extended be- 

 yond 15-20 minutes, it is advisable to cool the substrate mix- 

 ture with ice and to employ the lowest possible pH value 

 compatible with good enzymatic activity and prompt azo- 

 coupling. If longer incubation is carried out, the entire mix- 

 ture should be renewed every 30 minutes or so. 



Table 1 lists, diazonium compounds which have been found 

 to be useful because they give intense shades and are rela- 

 tively stable. 



The azo dyes formed from tetrazotized dianisidine and a- 

 naphthol are insoluble in alcohol or xylol (but soluble in a 

 mixture of the two; blot slide carefully between the last al- 

 cohol and xylene ) and can be mounted in balsam. All other 

 azo dyes formed from the ingredients mentioned are more or 

 less soluble in absolute alcohol and especially in xylene. They 

 must be mounted in glycerol- jelly or some similar medium. 



As mentioned, azo-coupling either does not take place at 

 all or is sluggish below pH 6. The pictures obtained, even in 

 the best case, lack precision of detail, and centers of activity 



