Changes in the Density of Cytochrome P-450 2B1/2 Molecules in the Membrane of Endoplasmic Reticulum in Perivenular, Midzonal and Periportal Hepatocytes of Rats after Administration of Phenobarbital.

Amplification of Immunostaining Intensity by the Peroxidase-Antiperoxidase, Avidin-Biotin-Peroxidase Complex or Catalyzed Signal Amplification Method Gives Erroneous Antigen Content in Sections.

“…Under the incubation conditions, effects of antibody trapping and steric hindrance are shown to be negligible in the sections [9,18]. The sections were washed with PBS and mounted in glycerol.…”

“…For image analysis, sections were examined in a TMD microscope (Nikon, Tokyo, Japan) equipped with an IF-470 interference filter (Nikon) and linked to an ARGUS 50 video image processor (Hamamatsu Photonics, Hamamatsu, Japan) as described previously [15][16][17][18]. Specific average absorbance in the widely defined areas in the sections (0.98 x 0.98 mm; ASIspec) and specific absorbance in small portions in hepatocyte cytoplasm (1 x 1 jim SIPspec) were calculated by subtracting absorbance in sections incubated with normal serum from that of sections incubated with the primary antibody.…”

“…7B, C). The extinction coefficient of the IPO method was calculated to be 300 [9,18]. Thus, CYP2B1/2 content in hepatocyte cytoplasm of PB-treated animals was 93 ± 7.4 (nmoles/cm3 cytoplasm; means + S.E.…”

“…Immunostaining intensity of cytochromes P-450 2B1/2 (CYP2B 1 /2) stained by using the indirect immunoperoxidase (IPO) method under saturation conditions is shown to be proportional to the content of the enzymes in sections [9,16,18]. Thus, molar content of the enzymes can be measured by quantitative immunohistochemistry [9,18] .…”

“…Thus, molar content of the enzymes can be measured by quantitative immunohistochemistry [9,18] . However, a sensitive immunohistochemical method is necessary to measure small amounts of antigens in sections by quantitative immunohistochemistry, because the sensitivity of the IPO method is low [16].…”