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about 2.5 mg antibody/ml as adjudged by quantitative precipitation.
Rabbit antiserum to mouse IgM (Ra-M IgM) was kindly provided by Mr.
Alan Brown, University of Florida (17) and contained antibodies against
mouse y and k chains. Fluorescein labeled goat antibody against rab
bit IgG was purchased from Miles Laboratories.
Bluegill serum immunoglobulins (Ig's) were purified from whole
serum by affinity chomatography, as discussed under Results, using
Ra-BIg coupled to Sepharose 4B by the CNBr technique (39). Elution of
adherent proteins was accomplished with 0.5 M acetic acid, 1.5 M NaCl.
Mouse IgM was kindly provided by Mr. Alan Brown, University of Florida,
after purificantion, as described previously (17), from the serum of
Ascaris infected mice. The extinction coefficient of bluegill and mouse
proteins (E28O cm) was assumec^ t0 be 13.5.
Preparation of Lymphocytes
The preparation of organ cell suspensions, isolation of leucocytes
from heparinized blood and organ cell suspensions and characterization
of leukocyte isolates have been described in Chapter II.
Membrane Immunofluorescence Studies
n
Bluegill lymphocytes were suspended at 2 x 10 cells/ml in ice
cold phosphate buffered saline containing 5% fetal calf serum (PBS-
FCS). One tenth ml aliquots were incubated for 30 min on ice with an
equal volume of various dilutions of rabbit sera (normal or Ra-BIg)
and washed three times with ice cold PBS-FCS; in order to inhibit
"capping" the washing solution used contained 0.03 M sodium azide.
The washed cells were then suspended in 0.2 ml of cold fluorescein
conjugated goat anti-rabbit IgG in PBS-FCS (with or without azide