124 



MACROMOLECULAR COMPLEXES 



Fig. 4. Cryofixation assembly for semiautomatic freeze-substitution, infiltra- 

 tion of specimens, and photopolymerization embedding. After rapid freezing 

 in liquid helium 11 or Freon 22-liquid nitrogen, the specimens remain in special 

 chambers (arrow) within refrigeration unit (at —130° to —90° C), emerging 

 ready for sectioning. 



prepared in anhydrous (less than ten parts per milHon water con- 

 tent ) form by passage through Linde 4A molecular sieves, followed 

 by ultrafiltration to remove suspended particles. Protective treat- 

 ment (Fernandez-Moran, 1952; Lovelock, 1953; Parkes, 1951) by 

 controlled glycerination (30 to 60 per cent glycerol-veronal buffer 

 solutions) of larger tissue specimens prior to freezing, with subse- 

 quent low-temperature transfer over alcohol or methylcellosolve 

 mixtures to the acrylic monomers, was found to be essential for 

 adequate preservation of fine structure (Fernandez-Moran, 1959a, 

 1959c). Osmium cryofixation used mainly in this study involved 

 ( 1 ) freezing of the tissues in liquid helium II ( or in Freon 22-liquid 

 nitrogen at —150° C for control purposes); (2) freeze-substitution 

 at —130° to —80° C in 2 per cent osmium tetroxide in acetone-ethyl 

 chloride mixtures; (3) impregnation at — 75° C in mixtures of 

 methyl acrylate and butyl-methyl methacrylate monomer; and (4) 



