5-AF594 tyramide is a red fluorescent dye (Ex=590 nm, Em=618 nm). It serves as a fluorescent substrate reported for horseradish peroxidase (HRP)-catalyzed deposition, aiding in tyramide signal amplification (TSA). 5-AF594 tyramide is applicable in multicolor immunohistochemistry experiments (mIHC).

5-AF594 tyramide detection involves several steps: 1) Incubate slices in 0.25% Triton X-100 at room temperature for 10 minutes to permeabilize; 2) Incubate with primary antibody overnight at 4°C; 3) Block endogenous peroxidase activity by incubating in 1x PBS with 3% H2O2 for 20 minutes; 4) Incubate with FITC-conjugated anti-rabbit IgG primary antibody at room temperature for 1 hour (1:100 dilution), followed by incubation with FITC-conjugated HRP antibody at 4°C overnight (1:500 dilution); 5) Incubate with 5-AF594 tyramide for 10 minutes (1:200 dilution); 6) Detect using a fluorescence microscope. Note: It is recommended to determine the optimal concentration of 5-AF594 tyramide working solution based on specific conditions.