Alexa 594 conjugated anti rabbit igg

Dual FISH was performed on sections from three adult rats, by mixing the digoxigenin-labeled Prss56 and fluorescein-labeled Tshb probes. Following hybridization, sections were first incubated in the peroxidase-conjugated sheep anti-digoxigenin antibody, and the signal was amplified using the TSA Plus DIG Kit (Cat# NEL748E001KT, Perkin Elmer) for 30 min, applying the DIG amplification reagent at 1:500 dilution in 0.05M Tris (pH 7.6) containing 0.01% H₂O₂. Sections were then incubated in a rabbit monoclonal antibody against digoxigenin (Thermo Fisher, Cat# 700772, RRID:AB_2532342; at 1 μg/ml concentration) for 3h, in the presence of 2% sodium azide to inactivate peroxidase activity. Sections were thoroughly washed in PBS, and incubated overnight in peroxidase-conjugated sheep anti-fluorescein antibody (Roche, Cat# 11426346910, RRID:AB_840257; diluted 1:100 in 1% blocking reagent). Signal amplification was applied using the TSA Plus Biotin Kit as described above, and the signals were detected with the cocktail of Alexa Fluor 488-conjugated Streptavidin and Alexa 594-conjugated anti-rabbit IgG (Jackson; 1:200). The red and green fluorescence of Alexa 594 and Alexa 488, respectively, were swapped in the images to keep the green color consistent for Prss56.