338 Histochemistry (chap. 21) 



might be damaged by the denaturing action of the alcohol as it warms 

 up. 



procedure: (Feeler and Sidman, 1938; Hancox, 19^7 ; Patton and Brown, 

 1958). 



1. Cool a beaker of liquid propane-isopentane (3:1) to — 170°C- 

 175°C with liquid nitrogen. 



2. Place tissue specimen (small pieces, 1.5-3 mm.) on thin strip of 

 aluminum foil and plimge into the cold propane-isopentane 

 (quenching). Stir vigorously with a 12" ruler: 30 seconds. 



3. Transfer frozen specimen to ice-solvent (see Comments) (already 

 chilled to —60 to — 70°C). After a few minutes the tissue can be 

 shaken loose from the foil. Store in dry ice chest for 1 to 2 weeks. 

 (52 days did no harm; Patten and Brown) 



4. Remove fluid and tissue to refrigerator, wash 12 hours or longer 

 in 3 changes of absolute alcohol. 



5. Transfer to chloroform: 6-12 hours in refrioerator, to remove 

 any traces of water. (This step may be omitted in most cases.) 



6. Transfer to cold xylene (or similar agent), — 20°C; and remove 

 to room temperature for 10 minutes. 



7. Transfer to fresh xylene, room temperature: 10 minutes. 



8. Transfer to xylene: tissuemat, 56°C, vacuum: 15 minutes. 



9. Infiltrate, tissuemat #1, vacuum: 15 minutes. 



10. Transfer to tissuemat #2, vacuum: 15 minutes. 



11. Transfer to tissuemat #3, vacuum: 15 minutes. 



12. Embed. 



comments: 



Ice-solvents used with best results by Patten ajid Brown {1958) were 

 absolute methyl and absolute ethyl alcohol. Hancox (1957) used 

 rz-butyl alcohol and could infiltrate directly from the ice-solvent, 

 thereby bypassing the xylene step. Feder and Sidman (1958) used 1% 

 solution of mercuric chloride in ethyl alcohol, and osmic acid in 

 acetone, trying to combine a chemical and physical fixing action. The 

 latter authors include complete directions concerning obtaining, 

 storing and disposing of liquid nitrogen, isopentane and propane, 

 also details about equipment. 



Hancox (1957) obtained his best sections in a cryostat. He tried 

 dry mounting, 85% alcohol flotation and water flotation of sections. 

 The latter gave inferior results; certain cell inclusions had disap- 

 peared, and the cytoplasm resembled a vacuolated reticulinn. Patten 

 and Brown (1958) found that mounting on water gave the most dis- 



