6-AF488 tyramide

6-AF488 tyramide is a bright, green fluorescent dye (Ex=496 nm, Em=524 nm). 6-AF488 tyramide is utilized as reporter fluorescent substrate of horseradish peroxidase (HRP)-catalyzed deposition for tyramide signal amplification (TSA). 5-FITC tyramide can be used for multiplex immunohistochemistry (mIHC)^[1]. In Vitro: 6-AF488 tyramide assay^[1].
a. Place tissues in per well in a 24-well plate, and it’s important to not let tissues sections dry.
b. Permeabilize tissues with 0.2 % Triton X-100 in PBS for 5-10 min.
c. Rinse with PBS-T for 5-10 min, and then incubate tissues in 1 % H₂O₂ for 20 min to block endogenous peroxidase activity.
d. Rinse with PBS three times, 5-10 min each, and then incubate in 1 % blocking solution (4 % normal donkey serum + PBS-T) in PBS for 1 h for to block nonspecific binding sites.
e. Apply the primary antibody working solution for 2 h at RT or overnight at 4 °C, and then rinse with PBS three times, 5-10 min each.
f. Apply the HRP-conjugated secondary goat anti rabbit antibody (1:100 in 1 % blocking solution) for 2 h at RT , and then rinse with PBS three times, 5-10 min each.
g. Activate TSA buffer with 0.0015 % H₂O₂, and then incubate tissues in 6-AF488 tyramide working solution (1: 100 in the activated TSA buffer) for 5-10 min at RT.
h. Rinse with PBS three times, 5-10 min each, and then mount sections on slides.
i. Place in slide holder in a nearly vertical position for 10 min to dry slides, and then briefly rinse slides with ddH₂O following drying for 10 min.
j. Mount slides with mounting medium, we recommend using AntiFade Mounting Medium (with DAPI) (HY-K1047).
k. Fluorescence microscopy detection.
Note: it is suggested that the optimal concentration of 6-AF488 tyramide working solution according to actual conditions.