Rapid Antibody Conjugation with Fluorescent Dyes

The primary antibodies were conjugated with fluorescent dyes using a rapid conjugation kit (ab269823, Abcam). For each 10µL of primary antibody, 1µL of modifier reagent was added and mixed gently. The lyophilized powder was dissolved in 10µL PBS (D8537, Sigma-Aldrich), and 1µL of this solution was added to each antibody and mixed gently. The mixture was incubated at room temperature for 15 min in the dark. After incubation, 1µL of quencher reagent was added for each 10µL of antibody used and was then gently mixed. In this experiment, the antibodies were conjugated to Alexa Fluor 647, which is a bright dye with less background fluorescence^{25 (link)}. While Alexa Fluor 488 is the brightest among the dyes, the channel has higher background fluorescence. Alexa Fluor 555 is the weakest dye, suitable for staining proteins with high abundance and high affinity.