AUTORADIOGRAPHY 329 



entirely satisfactory for cytoplasmic fixation (18). Bouin's solution has 

 been reported as satisfactory by Fitzgerald et al. (10), but it produced 

 some uncertainties in the hands of Doniach and Pelc (18). 



The extent of leaching depends upon the radioisotope and its combina- 

 tion in the tissue. For example, inorganic P*^ in a tissue is easily leached, 

 whereas organically bound ?•'- is tenaciously retained. There are two 

 approaches to the leaching problem : the careful use of fixatives known to 

 be satisfactory for the specific samples, and the use of various freezing 

 methods in which there is no opportunity for leaching losses. Holt et al. 

 (19) found that loss of inorganic P^' from tissues was minimized by a fixing 

 procedure that called for 2 hr in alcohol-Formalin (9:1) and three changes 

 of dioxane (2 hr, 1 hr, 1 hr), followed by the usual paraffin infiltration and 

 embedding. The dioxane was shown to be preferable to the use of alco- 

 hols for dehydration. It is emphasized that Formalin should always be 

 neutralized, since acid fixatives tend to increase the leaching loss. For- 

 malin is usually neutralized by allowing it to stand over an excess of 

 magnesium carbonate. Blank et al. (IG) have described a procedure in 

 which losses were minimized by using cold propylene glycol as a fixative 

 and Carbowax as the embedding medium. Although paraffin is most 

 widely used as an embedding material, Carbowax may offer some advan- 

 tage in that it can be removed by aqueous solvents as well as fat solvents ; 

 when aqueous solvents are used, it is possible to retain fats in the tissue. 



Witten and Holmstrom (20) have described a freezing method that 

 avoids the use of liquids. The tissue specimen is frozen by means of CO 2 

 gas on the object disk of the freezing microtome and is then sectioned with 

 a microtome blade kept cold by dry ice held in place on its upper surface. 

 The section in the frozen state is transported directly from the blade to 

 the photographic emulsion. Holt and Warren (21) have described sim- 

 plified freeze-drying procedures for autoradiographic studies. Two 

 embedding media are compared — paraffin (Tissuemat, melting point 

 50 to 52°C) and Carbowax (three parts Carbowax 4000, three parts Car- 

 bowax 1000, and one part Carbowax 1500; melting point about 56°C). 

 The embedding material is placed at the bottom of the tissue tube, which 

 is attached to a commercial freeze-drying apparatus. The fresh tissue is 

 frozen at about — 170°C by immersion in isopentane cooled with liquid 

 nitrogen and quickly transferred to the tissue tubes. The tissue tubes are 

 held in Dewar flasks surrounded by dry ice and attached to the apparatus 

 for 48 to 82 hr at a pressure of about 10~' mm Hg. The dry ice is allowed 

 to evaporate, permitting a gradual warming to room temperature, and at 

 the end of the 48- to 72-hr period the tubes are warmed to melt the embed- 

 ding material and facilitate infiltration. The desired handling techniques 

 are then employed. 



If the autoradiographic preparation is such that the film and section 



