Simultaneous identification of a specific gene protein product and transcript using combined immunocytochemistry and in situ hybridization with non-radioactive probes.
Simultaneous identification of messenger RNA (mRNA) and proteins in the same cells or tissues is a valuable tool to help the cell biologist evaluate the cell secretory cycle. Some cells may produce the mRNA and delay the production of the proteins. Alternatively, the proteins may be rapidly secreted. Other cells may produce both in sequence within the same time frame. Because of this difference, some cells can only be identified by their mRNA product. Others may have both products. This presentation describes a non-radioactive approach to the detection of both products with dual-peroxidase labeling protocols in use in this laboratory since 1983. The first detection system uses biotinylated cRNA probes or oligoprobes in in situ hybridization along with antisera to biotin to detect the hybrid. The detection system is amplified by 2-3 layers of anti-biotin, second antibody (made against the anti-biotin) and streptavidin conjugated to horseradish peroxidase. After the mRNA is detected with a blue-black substrate (nickel intensified diaminobenzidine), the antigens are detected with immunoperoxidase techniques and orange-amber substrate. The in situ hybridization protocol can also be used at the electron microscopic level. Trouble shooting and control protocols are also described. This approach has been shown to be valuable for detection of pituitary hormones, growth factors mRNAs and antigens.
Childs
G V
GV
Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston 77555-1043, USA. gvchilds@utmb.edu
D000941:Antigens / Q000032:analysis*; D019921:Biotinylation; D002478:Cells, Cultured; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D008854:Microscopy, Electron; D010902:Pituitary Gland / Q000737:chemistry* / Q000166:cytology; D010907:Pituitary Hormones / Q000032:analysis*; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000032:analysis*
D000941:Antigens; D010907:Pituitary Hormones; D018075:RNA, Complementary; D012333:RNA, Messenger