In situ hybridization at the electron microscopic level using a bromodeoxyuridine labeled DNA probe.
A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM), and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.
Yao
C H
CH
Second Department of Pathology, Kobe University, School of Medicine, Japan.
Kitazawa
S
S
Fujimori
T
T
Maeda
S
S
D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine; D015342:DNA Probes; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate; D016259:Genes, myc; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D007942:Leukemia, Experimental / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000302:isolation & purification; D016707:Tissue Fixation; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D001973:Bromodeoxyuridine; D016650:Fluorescein-5-isothiocyanate