C -fos mRNA Northern blotting was performed according to Hisanaga and 
coworkers’ (1990b) method, which is a modification of the method of Auffray 
and Rougeon (1980). Following isolation of the RNAand electrophoresis, 
the RNA was transferred to nylon membranes by capillary blotting. C-fos 
riboprobe was prepared from full-length c -fos cDNA using SP6 RNA 
polymerase and 32 P-CTP. Radiolabeled RNA was purified over a Nensorb 
column. The nylon membranes were hybridized with riboprobe in 1 .5X SSC, 
50 percent formamide, 1 percent SDS gel, 0.5 percent nonfat dry milk, 0.5 mg/ 
mL denatured salmon sperm DNA, and 0.2 mg/mL yeast tRNA at 65 °C for 24 
to 36 hours. Membranes were washed twice at 65 °C for 30 minutes in 2X 
SSC 0.1 percent SDS, 0.5X SSC 0.1 percent SDS, 0.2X SSC 0.1 percent 
SSC. They were exposed to film at -70 °C with an intensifying screen. 
For detection of Fos protein and Fos-related antigen proteins, 
immunocytochemistry was performed. Neurons were fixed with 
pacaformaldehyde/lysine/polylysine fixative 2 hours after each stimulation. 
After two PBS washes, cultures were incubated for 20 minutes in PBS 
containing 2 percent goat serum, 0.2 percent Triton X-100, and 0.1 percent 
bovine serum albumin. Cultures were incubated with polyclonal antibody 
to Fos and Fos-like antigens diluted 1 :50 for 2 to 3 days at 4 °C. The first 
antibody was detected using a biotin-conjugated secondary antiserum 
(diluted 1 :200), followed by the avidin-peroxidase complex reacted with 
diaminobenzidine and hydrogen peroxide as previously described (Sharp 
et al. 1990; Sagar et al. 1988). 
Cultures stained for glial fibrillary acidic protein using monoclonal antibodies 
were negative, whereas 1 to 2 percent of the cells in each well were 
immunoreactive for vimentin (presumed glioblasts). The remaining 98 to 99 
percent of the cells stained for alpha-tubulin and neuron-specific enolase, 
demonstrating that the cultures were nearly purely neuronal. 
RESULTS 
Control cultures treated with saline, PBS, or vehicle showed that only a few 
neurons (0.5 to 1 .5 percent) stained for FLI, which was induced in a much 
greater percentage of neurons (6 to 10 percent) following treatment with 
glutamate, bFGF, zinc, VIP, K + , db-cAMP, and TPA. This occurred in 
magnesium-containing and magnesium-free medium. NGF, EGF, PDGF, 
somatostatin, NPY, and carbachol did not induce FLI in the cultured neurons. 
Glutamate, bFGF, zinc, VIP, K + , db-cAMP, and TPA induced c-fos mRNA in 
the cultured neurons. Pretreatment with NMDA receptor blockers prevented 
glutamate and db-cAMP induction of c-fos mRNA. Pretreatment with NMDA 
175 
