Enzymes 141 



6. Embed in paraffin under reduced pressure, as described 

 above. 



Steps 1-4 should be performed at icebox temperature. 



In the case of alcohol or acetone-alcohol fixation, steps 3 

 and 4 can be omitted; in step 5 benzene may be used instead 

 of chloroform. 



Sections are cut at 3-8 fx, floated on lukewarm water, 

 mounted with albumin-glycerol, and dried. The dried sec- 

 tions should be melted in the paraffin oven ( 5-10 minutes ) . 

 The coating of paraffin they acquire by this treatment ap- 

 pears to have a protective action against atmospheric influ- 

 ences ( oxygen, moisture ) . Unmelted sections may lose most 

 of thek activity in a matter of a few weeks or months, 

 whereas melted ones, just like uncut paraffin blocks, remain 

 virtually unchanged for many years. 



For use, the slides are carried through xylene and alcohols 

 to water. It may be advisable to protect the tissue against 

 loss of enzyme with a thin layer of collodion. This can be 

 done by flooding the slide (after the second alcohol) with 

 dilute ( about 0.5 per cent) collodion in alcohol-ether, shaking 

 off the excess, and hardening the membrane for a minute or 

 so in 80-95 per cent alcohol. Some substrates do not pass the 

 membrane readily. In such cases, protection with collodion 

 should be omitted, and even the collodion from the embed- 

 ding procedure should be removed by a short rinse in alco- 

 hol-ether or acetone. 



HISTOCHEMICAL REACTIONS FOR ENZYMES 



Sometimes the primary product of the reaction is an in- 

 soluble dye (e.g., red formazan from triphenyltetrazolium 

 chloride) or a soluble one (e.g., acid aniline dyes from the 

 corresponding leuco-dyes), permitting immediate visualiza- 

 tion of sites of activity. Soluble dyes, as a rule, are much less 

 reliable as far as correct localization is concerned, because 

 they will stain the true sites of liberation only if they have 

 an affinity to some structure present locally; otherwise they 

 may diffuse away and may even stain distant and inactive 

 structures for which they do possess an affinity. 



