Special Features of Histochemical Methods 11 



if the fixative is, applied in the form of a gas (formaldehyde 

 vapor) or if it contains a specific precipitant for the com- 

 pound investigated. The spontaneous diffusion of small mole- 

 cules, together with the violent drift of solutes, caused by the 

 difference in osmotic pressure (global or specific) between 

 tissue fluids and the fixative, will result in a more or less 

 marked distortion in the pattern of distribution of these 

 molecules within a short time. Applying the fixative by 

 means of vascular perfusion may eliminate gross displace- 

 ment but not the intracellular shift of solutes. 



Such a displacement of highly diffusible substances can be 

 prevented by the use of the freezing-drying technique, origi- 

 nally described by Altmann^ and perfected by Gersh.^ For 

 a detailed description of the apparatus and its use the reader 

 is referred to the bibliography;^— only the principles of the 

 procedure will be given here. 



Small pieces of fresh tissue are dropped into liquid air (or, 

 even better, into isopentane cooled by liquid air; tempera- 

 ture lower than —150° C. ) where they are frozen solid almost 

 instantly. Subsequently, they are dehydrated in vacuo at a 

 temperature around —30° C. This, dehydration may take from, 

 a few hours to weeks, depending on the temperature, the 

 efficiency of the vacuum, and the size of the tissue, etc. The 

 dehydrated tissue is then embedded in paraffin. The im- 

 portant point is that there is no liquid phase present at any 

 stage of the procedure; therefore, diffusion of solutes cannot 

 take place. The sections may be floated directly on the re- 

 agent (for instance, an alcoholic solution of silver nitrate 



1. Altmann, R.: Die Elementarorganismen und ihre Beziehungen zu den 

 Zellen (Leipzig: Veit & Co., 1890). 



2. Gersh, L: Anat. Rec, 53:309, 1932, and Bull. Interaat. A. M. Mus., 

 28:179, 1948. 



3. Hoerr, N. L.: Anat. Rec, 65:293, 1936; Hoerr, N. L., and Scott, G. H.: 

 Frozen-dehydration method for histologic fixation, in Glasser, Medical phys- 

 ics (Chicago: Year Book Publishers, 1944); Simpson, W. L.: Anat. Rec, 

 80:173, 1941; Packer, D. M., and Scott, G. H.: Bull. Intemat. A. M. Mus., 

 22:85, 1942; and Stowell, R. E.: Stain Technol., 26:105, 1951. 



