Tyramide signal amplification tsa

RNA FISH probe labeling and RNA FISH procedures were performed as described previously [96 (link)]. Biotinylated single-strand LAT RNA probe was prepared by in vitro transcription (Ambion) using plasmid pSLAT-2 as a template (gift from S. Efstathiou, University of Cambridge, UK). Biotinylated LacZ probe was prepared from the pCMV-LacZ plasmid (Clontech) using the nick-translation procedure (Invitrogen). Frozen sections were treated as described for DNA FISH up to the antigen-unmasking step using solutions containing 2 mM of the RNAse inhibitor ribonucleoside vanadyl complex. The sections were pre-hybridized in 50% formamide/2 × SSC and hybridized overnight with 60 ng of RNA probe in a 50% formamide buffer at 65°C for LAT and 37°C for LacZ. Sections were washed in 50% formamide/2 × SSC at 65°C, and in 2 × SSC at room temperature. Detection was performed using streptavidin-HRP conjugate, followed by Tyramide Signal Amplification (TSA, Invitrogen) with an Alexa Fluor 350- or 488-conjugated substrate, according to the manufacturer’s guidelines. The DNA-FISH procedure was performed starting from the methanol/acetic acid post-fixation step.