Simultaneous in situ detection of mRNA and apoptotic cells by combined hybridization and TUNEL

We established a new method to allow simultaneous in situ detection of mRNA expression and apoptotic DNA fragmentation in paraffin-embedded tissue sections. We used human thymic tissue to perform in situ hybridization with a digoxigenin-labeled CD95 (APO-1/Fas) ligand (CD95L)-specific probe followed by TdT-mediated biotin dUTP nick end-labeling (TUNEL) of apoptotic DNA fragments. Bound probes were visualized by an immunogold-silver enhancement technique and fragmented DNA was detected with a streptavidin-peroxidase system. This double labeling technique produced a distinct, dark cytoplasmic staining of CD95L mRNA-expressing cells and an intense red nuclear precipitate in apoptotic cells or bodies. This technique will be a useful tool for microtopographical analysis of apoptosis-related gene expression.