Immunoelectron Microscopic Labeling of Digestive Tract Stem Cells by Means of Bromodeoxyuridine Antibody.

Abstract

As an alternative method to 3H-thymidine radioautography, immunohistochemical staining against incorporated bromodeoxyuridine (BrdU), an analogue of thymidine, was performed on rat stomach and small intestine organs embedded in Lowicryl K4M resin. After the administration of BrdU to animals intraperitoneally, organ tissues were removed and processed in a routine manner for resin embedding procedures. Semithin and ultrathin serial sections were cut and incubated in mouse anti-BrdU monoclonal antibody and stained with anti-mouse IgG or protein A coupled with gold colloid. Semithin sections were further intensified by physical development using silver acetate as an ion donor. In the electron microscopic labeling, pretreatment with hydrogen peroxide increased the labeling intensity.The findings from both light and electron microscopic observation were compared and a good correlation was obtained. Various labeling patterns were found in S-phase cell nuclei from stomach and small intestines in vivo. Labeling patterns of nuclei matrix were divided into five subtypes according to labeling patterns at the electron microscopic level.

A New Immunocytochemical Technique for Ultrastructural Analysis of DNA Replication in Proliferating Cells After Application of Two Halogenated Deoxyuridines

“…However, only permeabilized cells incorporate biotin dUTP (Hozák et al 1994;Ferreira and Carmo-Fonseca 1997), which can give rise to an alteration of the fine structure of the cells. For this reason, bromodeoxyuridine (BrdU), a thymidine analogue that does not need cell permeabilization for its incorporation, is more commonly used (Thiry and Dombrowicz 1988;Raska et al 1991;Sobczak-Thepot et al 1993;Tsuyama et al 1994;Tamatani et al 1995).…”

Electron Microscopic Immunogold Labeling of Bromodeoxyuridine (BrdU) in Routine Electron Microscopy.

“…Attempts to avoid HCl treatment at the electron microscopic level have been reported on epon-embedded ultrathin sections of unosmicated cultured cells [13,18] and liver tissue [11], though HCl hydrolysis has been reported to be essential for the cryo-ultrathin sections and for the Lowicryl-and LR White-embedded ultrathin sections [14,17,[24][25][26]. We demonstrated here that HCl hydrolysis could be avoided in epoxy sections of specimens fixed with glutaraldehyde and osmiun tetroxide at the electron microscopic level.…”

“…Immunohistochemistry of BrdU required the combination of hydrolysis and proteolysis of tissues fixed with formaldehyde and glutaraldehyde [7,8,21,22]. Incorporated BrdU into DNA has been localized at the electron microscopic level by treatment of Lowicryl K4M-or LR White-embedded sections with high concentration of HCl [14,[24][25][26]. Attempts of omission of HCl hydrolysis have been employed to localize BrdU in culture cells embedded in epon [13,18], indicating that the HCl hydrolysis is required to demonstrate BrdU in tissue but not in culture cells at the electron microscopic level [14,26].…”