132
depending upon desirability of "capping"), incubated for another 30
min on ice, and washed three more times as above. Cytocentrifuge
(Shandon-Elliott, Inc.) slides were prepared (1-2 x 10^ cells/pellet),
air dried, fixed 1 min with ethanol, air dried and mounted under PBS
buffered glycerol (1:9). Cells (> 1000/slide) were examined using a
Leitz fluorescent microscope (E. Leitz Inc., Rockleigh, N.J.) with a
HBC 200W mercury bulb (Osrain, Berlin, Germany), a BG^ excitor filter
(Leitz) and a K490 barrier filter (Leitz).
Labeling, Extraction and Immunoprecipitation of Membrane Immunoglobulin
Bluegill lymphocytes (and mouse thymic and splenic lymphocytes
for comparative purposes) were surface radio-labeled (76) and then
lysed by treatment for 3 hr at room temperature with 0.5% Nonidet P40
(Shell Chemicals U. K. Limited, London, England) prepared.in 0.045 M
Tris-HCl, pH 8.5, 0.01 M EDTA. The lysates were centrifuged for 15 min
at 3000 rpm and the supernatent dialysed for 18 hr with stirring at 4C
against 100 volumes of buffered saline (0.14 M NaCl, 0.01 M Tris-HCl,
pH 7.4). The dialysed lysates were then centrifuged and immediately
subjected to indirect immunoprecipitation as follows. Various volumes
of the lysates (ranging from 250 yl to 1500 yl) were added to 25 yl
rabbit serum (Ra-BIg, Ra-GL chain or normal rabbit serum as a control)
and incubated at 37C for 30 min. Sheep anti-rabbit IgG at equivalence
(170 yl) was added and incubated at 37C for 30 min. Immune precipi
tates were allowed to form for 16 hr at 4C, thrice washed and then
counted for radioactivity. The same protocol was used with mouse
lymphocytes except rabbit anti-mouse IgM was used in the "sandwich."
Inhibition experiments involved the same protocol except that various