Worm Breeder's Gazette 7(2): 53
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
I have developed a procedure for staining C. elegans neuronal cells in the head. The procedure uses paraformaldehyde and proteinase K (as suggested by F. Schachat) and appears to overcome penetration problems in larger animals with minimal distortion of the relative positions of cells, which may facilitate the identification of the stained cells. 1) Prefixation with paraformaldehyde; a plate of young adults are washed off bacteria and incubated with 1ml of 2% paraformaldehyde in PBS in a test tube overnight at 0 C. 2) Proteinase K treatment; after washing the prefixed animals with M9 buffer several times, a portion is transferred onto a small agar plate. Twenty animals are picked and placed into a 5 l drop of proteinase K solution (2mg/ml, prepared by diluting stock solution of 10mg/ml enzyme per 1ml tris-Cl, 10mM, pH 8.0 with M9 buffer) on a microscope slide subbed with gelatin. (Gall and Pardue. 1971. Methods in Enzymology XXI pp.470-480.) The slide is kept in a humidified chamber ( an inverted agar plate) during the picking operation to minimize evaporation. The animals on the slide are subsequently cut behind the pharynx with a surgical blade. Again to minimize evaporation during the cutting operation, the slide is kept on a glass petri plate filled with ice-water. The incubation with proteinase K then continues in a humidified chamber for 15 to 35 min at room temperature. 3) Postfixation with methanol and acetone; slides are frozen and fixed as described for eggs (procedures 2) and 3) in WBG 7 (1) pp.73) except that solutions of alcohols for rehydration have been kept at -20 C prior to use and the 30% ethanol and subsequent PBS steps are omitted to minimize loss of specimens during the procedure. 4) Incubation with antibodies; after 50% ethanol step, the excess rehydration solution is wiped off the slide and 50-100 l of a BSA solution ( 10mg/ml in PBS) containing 1mM PMSF is added onto the specimens. The head portions are then transferred into a well on a multitest slide (8 well, Flow Laboratories) with a finely drawn pipette and rinsed with 20 l of the above solution 3 times (each for at least 15 min at room temperature). Antibody solution, 20 l, containing 0.2% saponin and 1mg/ml BSA is pipetted onto the head specimens and the slide is placed in a humidified chamber overnight at room temperature. The specimens are washed 3 times (each for 15 min at room temperature) with PBS containing 0.2% saponin and then incubated with FITC-conjugated second antibody solution containing 0.2% saponin for 1 hour at 37 C. It is then rinsed as before and examined with a fluorescence microscope. Some three quarters of the head specimens in a well can be stained more or less by this method using a monoclonal antibody obtained in our laboratory, which appears to recognize some specific neurones in the worm head (probably IL2). Step 1) is necessary to minimize distortion of the relative positions of the neuronal cells in the head during the next steps which are in turn required for penetration of antibodies.