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 sam e cells or tissues is a valuable tool to help the cell biologist evaluate t he cell secretory cycle. Some cells may produce the mRNA and delay the prod uction of the proteins. Alternatively, the proteins may be rapidly secreted . Other cells may produce both in sequence within the same time frame. Beca use of this difference, some cells can only be identified by their mRNA pro duct. Others may have both products. This presentation describes a nonradio active approach to the detection of both products with dual-peroxidase labe ling protocols in use in this laboratory since 1983. The first detection sy stem 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 t he mRNA is detected with a blue-black substrate (nickel intensified diamino benzidine), 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 detecti on of pituitary hormones, growth factors mRNAs and antigens.