160 MICROSCOPIC TKCHNIQUES 



PROCEDURE 



1. Prepare 10 /x })araffin sections and attach to slides with egg 

 albumin. 



2. Dry, deparaffinize with xylol, and carry through absolute 

 alcohol to 1% celloidin in alcohol-ether. 



3. Drain off excess celloidin by standing the slides in an empty 

 Coplin jar. 



4. Place in 70% alcohol for about 1 min. to harden the celloidin, 

 and dry at room temperature. 



5. In the dark room, pipette 5 drops of the melted photographic 

 emulsion on to each slide and spread evenly with a camel's hair brush. 

 Carry out this operation a little below 40° on a hot plate to prevent 

 premature gelling of the emulsion. (The temperature should be held 

 below 40° with this emulsion to reduce the fogging.) 



6. Allow to cool and dry, and place the slides horizontally in a 

 dustjiroof, light-tight box for the duration of the exposure. 



7. x\fter the exposure develop for 3-4 min. in Kodak developer 

 D72 at 18-20°. Wash rapidly in water and fix for about 10 min. in 

 5% thiosulfate at the same temperature. Wash finally in cold run- 

 ning water for about 30 min. The black silver deposit indicates the 

 site of the radioactive element. 



8. Counterstain in Coplin jars cooled in running water. (Methyl- 

 ene blue and Harris hematoxylin may be used for radiophosphorus 

 "autographs" and Harris hematoxylin for those of radioiodine.) 

 With methylene blue place slides in a 1% alkaline soln. for about 

 30 min. and rinse in running water until the stain is removed from 

 the gelatin coating. With Harris hematoxylin, stain lightly to avoid 

 the need of differentiating. (Artifacts caused by gelatin swelling and 

 disengagement of the sections disappear when the slides are thor- 

 oughly dried after each operation.) 



9. Pass through several changes of 95% alcohol, absolute alcohol, 

 and xylol, and mount in Canada balsam. (Clearing in oil of origanum 

 before mounting will also give good results.) 



Discussion 



Points on various procedures required for particular studies may 

 be obtained by referring to some of the applications already made. 

 To date, most of the investigations employing radioautography 



