NOTES ON TECHNIC

THE USE OF A FAST, COARSE-GRAIN STRIPPING FILM FOR RADIOAUTOGRAPHY

T h e design and use of fine-grain, permeable base stripping film for r ad ie autography has been reviewed recently by Pelc (1956).l A fast, coarse-grain film (AR50) similar in design to the fine-grain preparation (ARIO) has now become available from Kodak, Ltd., London, England. This film is designated Kodak Fast Autoradiographic Stripping Plate A.R. 50; the fine-grain prepa- ration is designated Kodak Fine-Grain Autoradiographic Stripping Plate A.R. 10. We have found this film useful for radioautographs requiring resolu- tion not better than 15-2Op and as a supplementary technic to the high reso- lution method. The film consists of an emulsion layer of 12p supported by a gelatin layer of lop. The developed grain size by our technics varies from 1 to 3p. The film is said by the manufacturer to be approximately 10 times as fast as the fine-grain emulsion. We have used AR50 stripping film satisfac- torily for the localization of iodine13l, phosphorus32 and zinc65 in various animal tissues. In several cases, activity could be demonstrated with this film in sections, when radioautographs of adjacent ones prepared with ARIO or NTB-2 emulsions were negative after maximum or long exposure times. When adequate activity is present and fine resolution is necessary, AR50 radio- autographs set u p simultaneously with those prepared with ARIO serve as an indicator of the distribution and intensity of the isotope and aid in the pre- diction of correct exposure times for the high resolution radioautographs.

Procedures for the preparation of radioautographs with AR50 are similar to those used with ARIO. Some improvements to previously published methods (Fitzgerald, Simmel, Weinstein and Martin, 19532 and Pelc, 1956) have been made and the technic used in our laboratory i s therefore given below:

1. Cool all solutions to 19°C. Develop 4 min in D-19; wash in distilled water. Fix for 15 min in a fixer diluted with 3 parts of distilled water. The stock fixer consists of 480 gm of sodium thiosulphate, 1000 ml of distilled water and 125 ml of Kodak liquid hardener. Wash for at least 0.5 hr in several changes of distilled water. The radioautographs may then be dried in a stream of cool air from a fan.

2. Cool all solutions to 18°C. If the radioautograph has been previously dried, soak it in distilled water for 30 min. Stain 5 sec in 0.25% Eosin Y (made LIP in 25% alcohol), wash in 2 changes of distilled water. Stain 1 hr in Erlich's acid hematoxylin. Wash briefly, destain about 45 sec in 0.2% HCl. Blue in running tap water for at least 30 min. Fan-dry the stained radioautograph.

'Pelc, S. R. 1956. The stripping film technique of autoradiography. Internatl. J. Appl.

*Fitzgerald. P. J.. Simmel, E., Weinstein, J., and Martin, C. 1953. Radioautography: Radiation and Isotopes, 1 , 172-7.

theory, technic, and applications. Lab. Investig., 2, 181-222.

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300 STAIN TECHNOLOGY

3. Place the radioautograph for 30 min in a solution of Akriloid B-72 Rohm

Prepare a 40% solution of the polymer in xylene; place it in the paraffin oven for several days to dissolve it. T o 1 part of this solution add 1 part of chloroform.

Remove the radioautograph from the Akriloid B-72 solution, allow to drain and dry overnight in a desiccator containing silica gel by standing the slide on edge on some filter paper. Radioautographs thus coated with B-72 have been well preserved in this laboratory for over a year. Xylene will dissolve the coating, hence oil used for oil-immersion magnification must be removed with dry lens paper. Such preparations are particularly suitable for low resolution radio- autographs where oil-immersion magnification is not needed.-Eva B. SIMMEL, Zliitision of Biophysics, Sloan-Kettering Institute, h'ew York, New York.

and Haas, Philadelphia) made up as follows:

A METHOD FOR EMBEDDING SMALL SPECIMENS~

During the course of a histochemical study on the slime mold, DictyosteEium discoideum Raper (size range: 0.3-3.0 mm), we devised a method for processing whereby any single individual could be properly oriented for serial sectioning. Previously, individuals were harvested as a mass of perhaps as many as 100 organisms, with a consequent random orientation and with the chances of obtaining a desired plane of sectioning greatly reduced. In the method described here, the desired plane of sectioning is consistently achieved.

Dictyostelium discoideum is cultured on a nutrient agar medium in Petri plates. Initial fixation of the tissue is accomplished by flooding the entire plate with ice-cold acetone. Under a dissecting microscope, the desired stages of the life cycle are then picked off with watchmaker's forceps and transferred to a small Stender dish, also containing ice-cold acetone. Complete fixation in this medium is accomplished within 2 hr.

After fixation, the several individuals, still in one Stender dish, are passed through the various reagents leading to embedding. The fluids are changed with a fine-tipped pipette while the Stender dish is held on a slant.

T o introduce a specimen into melted paraffin, it is picked up with forceps from the fluid (e.g., benzene) in the Stender dish. The forceps are immediately wiped dry with absorbent tissue, except for the tip holding the organism, to prevent excess solvent being carried over into the paraffin. The forceps and tissue are then introduced into the embedding dish of melted paraffin. The paraffin in immediate contact with the forceps solidifies, forming a semi-solid

'Developed during the tenure of grants from the National Science Foundation (NSF G- 2617) and the Tulane University Council on Research.

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