75
from total mean DPMs of triplicate or quadruplicate
coverslips. Nonspecific background DPMs were obtained by-
incubating unpurified cells in an irrelevant monoclonal
antibody specific for pipefish vitelline envelope (provided
by Dr. P.C.Begovac, Whitney Laboratory) for 30 min. on ice
and then processing them identically as those incubated in
A2B5.
Immunohistochemistrv
All immunohistochemistry of cells and monolayer
cultures was performed as described in the previous chapter
except as noted below. 5A11 (Linser and Perkins, 1987b)
immunohistochemistry of monolayers was carried out
identically to that for A2B5. Immunostaining of cells for
glutamine synthetase was accomplished by permeablization
after formaldehyde fixing with 95% ethanol for 2 min. at
-20C, rinsing, and incubation in a 1/100 dilution of
polyclonal anti-GS (Linser and Moscona, 1979) for 30 min.
Other NF antibodies rasied against rat neurofilaments and a
monoclonal antibody specific for GFAP (DA3, NN18, anti-MSH,
A5; kindly provided by Dr. G. Shaw, Univ. of Florida) were
also used to immunostain monolayers.
3H-Thvmidine Incorporation
New DNA synthesis in cell cultures was investigated by
3H-thymidine autoradiography combined with immunohisto
chemistry as described in the previous chapter.