Tyramide signal amplification kit tsa

PCR products from embryonic cDNA for the vg gene or the vg PRE/TRE ncRNA were cloned in both orientations into PCR XL Topo (Invitrogen). For in situ hybridization, RNA probes were in vitro transcribed from each cDNA strand. One probe was Dig labeled (Boehringer), detected with sheep Anti-Digoxigenin-AP (Roche) and visualized with FastRed (Sigma). The other probe was labeled with fluorescein and detected with primary antibody: Mouse anti-fluorescein (Roche), secondary antibody: goat anti-mouse-HRP (Invitrogen) and visualized with Alexa Fluor 488 Tyramide (Invitrogen) using Tyramide Signal Amplification kit (TSA™, Invitrogen) according to manufacturers instructions. Label swaps were performed to ensure specificity. In the images shown, vg mRNA was detected with Fast Red, and PRE/TRE transcripts were detected with TSA. Images were taken using confocal microscopy, with LSM 700 Axioimager (larval tissues) or LSM 510 Axiovert 200M (embryos) (Zeiss).