The utility of in situ detection, including RT in situ PCR, of viral nucleic acid and the co-localization of the cytokine response to the study of viral pathogenesis

This manuscript focuses on the detection of viral nucleic acids by in situ based methodologies. The optimal protocol depends on the virus. We will describe protocols for viral RNA detection by reverse transcriptase (RT) in situ PCR. We will also directly compare this method to the detection of viral RNA using standard in situ hybridization with locked nucleic acid (LNA) probes. Most DNA viruses are associated with high viral copy number and, thus, can be detected by standard in situ hybridization. Retroviral provirus is an exception as the single integrated DNA is best detected by PCR in situ hybridization. We will also describe protocols for the co-localization of viral DNA and RNA with host cytokines. Our protocol typically has the protein immunohistochemistry as the second step, with the key features being pretreatment step, antibody concentration, co-labeling analyses with a computer-based system, and co-analyzes with serial sections.