Immunohistochemical detection of proliferating lipocytes in regenerating rat liver

Abstract

For the detection of proliferating lipocytes in regenerating liver, partially hepatectomized rats were injected intrapcritoneally with 5‐bromo‐2′‐deoxyuridine (BrdUrd, 50 mg/kg body weight) and killed 1 h later. Acetone‐fixed frozen liver sections were used for the simultaneous detection of cytoplasmic desmin and BrdUrd‐labelled nuclei of lipocytes using double immunohistochemical procedures. The best results were obtained with the sequences: rabbit anti‐desmin → biotinylated anti‐rabbit IgG → avidin‐biotin‐peroxidase complex → 3,3′‐diaminobenzidine tetrahydrochloride, followed by DNA denaturation → mouse anti‐BrdUrd → anti‐mouse IgG → peroxidase‐anti‐peroxidase complex → 4‐chloro‐l‐naphihol. With this method, cytoplasmic desmin was stained a brown colour, which sharply contrasted with the blue‐stained BrdUrd‐labelled nuclei. Uniabelled nuclei appeared green after counterstaining with methyl green. No cross‐reaction between immunoreagents of desmin and BrdUrd stainings was observed. The labelling index of lipocytes peaked (25.7 percent) 48 h after partial hepatectomy, whereas it was 3.7 per cent in normal rat liver.