Vimentin immunohistochemistry in human fetal brain: methods of standard incubation versus thermal intensification achieve different objectives.

Abstract

Vimentin is transiently expressed in many cells of neuroectodermal origin in the fetal central nervous system and may be demonstrated by immunohistochemistry in paraffin sections. Traditional methods incubate antibodies with tissue sections at room temperature, but boiling the incubating solution in a microwave oven enhances antigen retrieval and greatly intensifies immunoreactivity. This study compares vimentin immunoreactivity using various dilutions of a commercial monoclonal antibody and various incubation conditions including room temperature, overnight at 4 degrees C, and after heating to boiling either in a microwave oven or on a hot plate. Fifteen fetal brains and spinal cords were examined, ranging in age from 8 to 24 wk gestation. Normal brains of four term or near-term neonates and two with hypoxic/ischemic lesions were also studied. Hot plate and microwave heating were equivalent. No differences in immunoreactivity could be attributed to gestational age per se. Boiling for 10 min in a microwave oven at a dilution of 1:200 is recommended for the enhanced demonstration of fetal histological structures such as radial glial fibers, but it is not recommended for studies of maturational gradients of cellular vimentin that differ at each gestational age, as in fetal ependymal cells or during the conversion of radial glia to mature astrocytes. For semiquantitative developmental studies, vimentin incubation at room temperature or overnight in the refrigerator is recommended at a dilution of 1:25 or 1:50 to avoid misinterpretation of apparent excessive staining after thermal intensification. Recommendations of commercial suppliers focus on the identification of neoplastic cells and must be modified to demonstrate subtle developmental changes in fetal tissues.