Antigen retrieval by microwave oven heating for immunohistochemical analysis of bone marrow trephine biopsies.

Immunohistochemical analysis of bone marrow trephine (BMT) biopsies with monoclonal antibodies for the analysis of hemopoietic disorders has been hindered by the fixation and decalcification regimens which mask or destroy tissue antigens. This study evaluated the effect of microwave oven treatment on the quality of immunostaining of fixed decalcified trephine biopsies. The aim was to establish whether this method of pre-treatment would enable additional antigens to be detected. Fifty-eight monoclonal and 4 polyclonal antibodies to hemopoietic antigens were assessed to compare no tissue pre-treatment, proteolytic (trypsin) enzyme digestion and microwave oven heating. The microwave heating of the sections was performed by placing them in a boiling solution of 0.01M tri-sodium citrate for a total of 10 mins. Following microwave heating 14 antibodies that previously showed no reactivity in BMT biopsies gave positive staining and 9 antibodies previously known to detect antigens in the absence of pre-treatment gave enhanced staining. Other antibodies showed no staining improvement with microwave heating and some failed to give a positive reaction by any of the pre-treatment methods. Antigen retrieval utilizing microwave oven heating can expose antigenic sites for antibody binding in bone marrow trephine sections. However not all antigens are retrieved and there is variation between epitopes on the one molecule and their ability to be exposed by microwave heating. Utilizing antigen retrieval methods, the range of antibodies applicable to BMT sections is greatly expanded enabling the immunophenotypic analysis of the majority of hemopoietic disorders.

Independent mechanisms for tumor promoters phenobarbital and 12-O-tetradecanoylphorbol-13-acetate in reduction of epidermal growth factor binding by rat hepatocytes.

Primary cultures of hepatocytes derived from adult Fischer 344 rats were used to test for effects of the liver tumor promoter phenobarbital on several components of the epidermal growth factor (EGF) receptor signal transduction pathway. Phenobarbital had no effect on the binding of 125I-labeled EGF by its hepatocyte receptor at 4 degrees C or on EGF-induced receptor down-regulation. However, pretreatment of hepatocytes with phenobarbital (3 mM) at 37 degrees C caused inhibition of subsequent 125I-labeled EGF binding. This response temporally resembled that of hepatocytes to 12-O-tetradecanoylphorbol-13-acetate (TPA) in that maximal inhibition occurred after 1 h of pretreatment but was reversed after longer pretreatment times. The inhibitory effects of phenobarbital and TPA on EGF binding were additive, suggesting that distinct mechanisms mediated the responses to these two tumor promoters. In addition, treatment with TPA, but not phenobarbital, caused a redistribution of the activity of Ca2+/phospholipid-dependent protein kinase C. In untreated and phenobarbital-treated hepatocytes, 20% of protein kinase C activity was isolated with a membranous fraction, while 75% of the activity was membrane associated in TPA-treated hepatocytes. These results demonstrate that phenobarbital, like TPA and other tumor promoters, can modulate the EGF receptor system but suggest that it does so without directly competing with EGF for binding to its receptor or by activating protein kinase C.