Correlation of fixation, HCL hydrolysis and proteolytic digestion in bromodeoxyuridine immunohistochemistry of human stomach labeled by extracorporial perfusion system.

Abstract

Bromodeoxyuridine (BrdU) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. To determine the optimal conditions for detecting BrdU-incorporated nuclear DNA in formalin- or ethanol-fixed, paraffin-embedded human gastric tissues, we tested several different pretreatment procedures of hydrolysis with HCl and enzymatic digestion with actinase before the immunohistochemical staining. Three cases of surgically resected stomachs were perfused with artificial blood containing BrdU and fixed in formalin, ethanol or with their combination for different durations. The results of BrdU-immunohistochemical stainings were checked and the labeling indices were compared. As for hydrolysis, treatment with 2N HCl at 25°C for 90min was sufficient to detect the immunoreactivity in the stomach irrespective of fixative conditions tested. However, to obtain the most suitable stainings an additional enzymatic digestion was needed, and the optimal condition with actinase was different according to the fixation time in formalin- and/or ethanol-fixed tissues. There are also a few differences in the demonstrability of nuclear BrdU among various kinds of cells, i.e. normal epithelial cells, immunoblasts in the lymphoid follicle and cancer cells. Our method could extend the range of application of BrdU immunohistochemistry for cell kinetic studies using human surgically resected organs.