An Improved Method for EM Radioautographic Techniques using Cork

EM Radioautographic Techniques에 관(關)한 연구(硏究) - Cork 방법(方法) -

Electron microscope radioautography introduced by Liquier-Milward (1956) is now used routinely in many laboratories. Most of the technical difficulties in specimen preparation have been overcome. This method is modified from loop method for improvement of EM radioautographic techniques. The advantages of this method are: 1. the use of single specimens on small corks and of a large wire loop, allows the experimenter to avoid the blemishes in the membrane; 2. the surfactant dioctyl sodium sulphosuccinate is added to diluted ILford L4, thus greatly prolonging the period of time over which good emulsion layers can be made; 3. corks can be handled in perspex holder which allows about 20 specimens to be developed simultaneously. The steps of the method comprise: 1. Cut ribbons of ultrathin sections of silver interference colour 2. Pick them up on formvar-coated 200 mesh grids 3. Prestaining of tissues 4. Coat the specimens with a thin layer of carbon by evaporation (30-60A) 5. Mount the specimens on corks (about 1cm apical diameter) using double-sided scotch tape 6. Emulsion coating; a. Take a 250m1 beaker, place it on the pan of a sliding weight balance and weigh it. Add 10 grams extra to the beam. Add pieces of ILford L4 emulsion to the beaker until the balance is swinging freely. Add the 20ml of distilled water that was previously measured out. b. Surfactant dioctyl sodium sulphosuccinate is added to diluted ILford L4. 7. Prepare a series of membranes of gelled emulsion with the wire loop and apply one to each cork-borne specimen. 8. Put the specimens away to expose by pushing the corks into short length of PVC tubing, each tube having a small hole in the side 9. Place the tubes in small boxes together with silica gel. 10. Exposure 11. Developer - Kodak Microdol X for 3 minutes 12. Fixer - A perspex holder can be manufactured which allows 20 specimens to be developed simultaneously. 12. Fixer - 30% sodium thiosulfate for 10 minutes 13. Examination with Siemens Elmiskop 1A electron microscope