340 Histochemistry (chap. 21) 



4. Infiltrate with dilute celloidin or nitrocellulose (approximately 

 10%): overnight, refrigerator. 



5. Drain on cleansing tissue. Place in benzene or chloroform: 0.5-1 

 hour. Stirring on a mag-mix aids penetration of fluid. 



6. Infiltrate with paraffin, 56°C (52°C is better): 15-20 minutes in 

 oven without vacuum. 



7. Infiltrate with paraffin, 56°C (or 52°C): 30 minutes with vacuum. 



8. Infiltrate with paraffin, 56°C (or 52°C): 10-15 minutes without 

 vacuum. 



9. Embed. Store in refrigerator. 



10. Section. Float on lukewarm water, briefly (10 minutes maximum). 

 Drain off water and dry. If kept for some time, put in oven 5-10 

 minutes to melt paraffin forming protective coat against atmos- 

 pheric influence. Store in cold room or refrigerator. Safest ^vay 

 to store tissues is in uncut paraffin block in refrigerator. 



comments: 



Novikoff et al. (1960) find acetone fixation is good for some cryostat 

 preparations. 



Control Slides: Most enzyme reactions should be accompanied by a 

 control slide in order to help detect false positives. An enzyme can be 

 inactivated by one of the following methods: 



1. Place slide in distilled water and bring to boil: 10 minutes. 



2. Immerse in IN HCl: 15 minutes; wash in tap Avater: 0.5 hour. 



3. Incubate slide in substrate medium containing an inhibitor. 

 NaCN or KCN. 



4. Incubate untreated slide in a medium backing substrate. 



Preservation of incubation media: Klionsky and Marcoux {I960) 

 prepared twelve different media, froze them in dry ice-acetone mix- 

 ture, and stored them in a dry ice chest. Within at least 6 months, the 

 solutions remained as good as freshly prepared solutions. The types 

 of media tested were: 



1. azo dye and metal precipitate methods for acid and alkaline phos- 

 photases. 



2. alpha-naphthyl and indoxyl acetate for esterase. 



3. 5-nucleotidase. 



4. nitro-BT for succinic dehydrogenase. 



5. post-coupling technic for beta-glucuronidase. 



6. triphosphopyridine nucleotide and diphosphopyridine nucleotide 

 diaphorase. 



