Immunocytochemical localization of RasHa p21 in normal and neoplastic cells in fixed tissue sections from Harvey...

An affinity purified sheep IgG antibody to a 20 amino acid peptide from the carboxyterminal end of RasHa p21 was used to localize RasHa p21 on fixed tissue sections of Harvey sarcoma (HaSV) virus-infected mice by the avidin-biotin-peroxidase immunocytochemical technique. Control sera included immune sheep sera absorbed with the peptide, preimmune sheep sera and a goat polyclonal antibody to Rauscher leukemia virus p30. Neonatal BALB/c mice were injected with HaSV/Moloney leukemia virus (MoLV), MoLV alone or buffer. Short-term fixation in Bouin's fixative was found to be the most effective method for demonstrating p21 in fixed tissue sections. RasHa p21 was found in 5-80% of the induced sarcoma cells, depending on the tissue fixative and antibody dilution. The antigen was localized to the cell membrane and in the cytoplasm. Tumors induced by NIH 3T3 cells transformed with cellular Ha-ras oncogenes had less than 1% immunoreactive tumor cells. Splenic erythroblasts in HaSV-induced erythroblastosis contained membrane antigen as did some reticular cells in lymph nodes draining the sarcomas. Normal tissues of virus-inoculated mice, uninoculated controls or fetuses and selected naturally occurring or induced liver tumors of mice, chemically induced skin tumors of mice, N-nitrosomethylurea-induced mammary tumors of rats, and naturally occurring tumors of F344/NCr rats did not contain immunoreactive p21. Thus, with the use of affinity purified IgG sheep polyclonal antibody to a peptide in RasHa p21, we were able to demonstrate RasHa p21 in tumors and other cells. The degree of immunoreactivity was related to the expected level of p21 expression.