Laurel J. Karr

In situ hybridization for cytokine mRNA with digoxigenin-labeled riboprobes. Sensitivity of detection and double label applications

A sensitive in situ hybridization procedure using both digoxigenin and 35S-labeled riboprobes is described that allows detection of single T cells expressing cytokine mRNA species in both single and double label formats. Modifications to existing procedures have been developed that allow in situ hybridization to be performed in either fresh frozen tissue sections or cytocentrifuge preparations of cultured cells. For single label studies, the digoxigenin labeling technique is equivalent to 35S labeling for sensitivity of detection and is superior with respect to precise localization and ease of use. A procedure to detect two cytokine mRNA species in individual cells can be performed using one digoxigenin-labeled riboprobe and one 35S-riboprobe, with equivalent sensitivity between the two labels and no non-specific mixing of the two signals. Since production of many T cell cytokines are controlled by transcriptional mechanisms, the use of in situ hybridization will be useful to investigate the biology of T cell activation, patterns of cytokine phenotype development, and histological localization of cytokine expressing cells in inflammatory lesions. Initial studies using this method to examine cytokine expression by a panel of T cell clones reveals that individual cytokine genes are not necessarily expressed in coordination in individual cells and relatively few individual cells in a Th0 clone express Th1-like and Th2-like cytokines simultaneously.