Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes

Abstract

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.