Silver-Impregnation Method of Peripheral Nerves with Frozen Sections

K. Kubota, one of the authors, has applied the following two methods of silver-impregnation : One is the Seto's method, introduced by Dr. Seto, Professor of Anatomy at the Tohoku University and the other is the Suzuki's method, modified by Dr. Suzuki, Professor of Anatomy at the Osaka City University. Based on these methods, the authors have accomplished their modification of staining peripheral nerves for frozen sections, which seems to be much easier for beginners than any other methods. Staining Procedures. 1. Fixation of material : Freshly isolated materials are fixed in 10% formol-alcohol for 10 days, then kept in 10-20 % formalin for a month. Hard tissues of teeth should be broken before fixation in order to fix the dental pulp. 2. Decalcification of hard tissues : After washing in running water, the formalin-fixed materials are immersed in the decalcifying solution of Morse (prepared by mixing equal amount of 20% solution of sodium citrate and 50% solution of formic acid) for a few days, changing the solution every day, until the specimens become rubber soft. 3. Again immerse in 10-20% formalin. 4. Cut frozen sections at 20-40 microns and keep in 10% formalin. 5. Pretreatment for staining : a. Immerse in neutral formalin for 7-14 days. b. After washing in distilled water, immerse in the mixture of 10 cc. of formic acid and 90 cc. of neutral formalin for about 4 days. c. After washing in distilled water, immerse overnight in the solution made up by mixing equal amount of absolute alcohol, neutral formalin and saturated solution of acid arsenous. d. After washing in distilled water, immerse in the mixture made up by mixing equal amount of 5% sodii bicarbonate solution, 15 % solution of hexaminum and neutral f ormal in for about 30 minutes. 6. Wash thoroughly in distilled water. 7. Immerse in 20% solution of silver nitrate for one -2 days at roomtemperature or for about 60 minutes at 37°C. 8. Wash rapidly in 10 % f ormalin, then wash quickly in distilled water two or three times. 9. After sucking up the water from the specimens with blotting paper, immerse in an ammoniacal silver solution (made up by adding 30% cold ammonia water drop by drop in 20 % solution of silver nitrate until dark brown precipitate completely dissolves) for about 10 minutes. Much better result was obtained by warming the solution slightly, for example, by immersing the container in a water bath. 10. Deoxidization of silver : a. Wash briefly in 10% cold solution of potassium sodi