146 Microscopic Histochemistry 



of the enzyme from sections of embedded material must be 

 considered unlikely/^- ^^ especially if collodion protection 

 is used. At least, solutions in which a larger number of slides, 

 carrying sections of highly active material, are soaked for 

 hours, and even days, never acquire any demonstrable activ- 

 ity. However, enzymes may be lost by diffusion from unfixed 

 or poorly fixed tissues (frozen-dried material ) unless the prop- 

 er precautions are taken (p. 12). The enzyme dissolved in 

 the incubating medium may slowly decompose the substrate, 

 cause its gradual saturation, and produce artifacts of the 

 type described in the preceding passage. Theoretically, the 

 enzyme itself may even be adsorbed in a secondary way on 

 various striictures; however, it has been shown experimen- 

 tally that such an adsorption can occur only under very ab- 

 normal conditions,^^ not in the least likely to be met with in 

 practice. 



In the case of precipitates which are not extremely insol- 

 uble, diffusion currents simply wash away part of the reac- 

 tion product and prevent its precipitation. On the other 

 hand, the relative sluggishness of these currents may cause 

 a somewhat different type of artifact if the precipitate is very 

 insoluble, mainly in the case of some azo dyes but possibly 

 even in the case of phosphates. Under such conditions the 

 reagent may become exhausted in the vicinity of centers of 

 very high activity more quickly than it can be supplied by 

 diffusion from near by. With this lack of precipitant, the 

 product of hydrolysis will remain unbound and free to dif- 

 fuse until it encounters a fresh supply of reagent some dis- 

 tance away. This will result in too little staining at the sites 

 of true activity and an irregular precipitate of azo dye (or, 

 in the case of Ca phosphate, staining of nuclei ) around them. 

 This type of artifact can be prevented by agitating the slide 

 vigorously during incubation, and so hastening the inter- 

 change of fluid around the tissue. 



18. Doyle, W. L.: Quantitative aspects of the histochemistry of phospha- 

 tases. In Symposium on cytology (East Lansing: Michigan State College 

 Press, 1951). 



