The purpose of secondary antibody staining is to bind to the primary antibody, thus amplifying the signal and allowing the spatial location of the primary antibody to be visualized through the emission of specific fluorophore signals in fluorescence microscopy.
Please note, the following procedure is a common protocol for adherent cells. In your specific case, please follow your own optimized and modified procedure to ensure the most accurate results.
Protocol
– After primary antibody staining and washing, prepare for secondary antibody staining.
– For a standard 18×18 mm cover glass, use 50 µl of secondary antibody solution.
– Remove the excess PBS and place the cover glass onto a drop of antibody solution, cells facing down.
– Create a humidity chamber to prevent the staining solution from evaporating. – Incubate for a common period of 1 hour at room temperature.
– After incubation, wash the cells three times with PBS, allowing the cells to remain in each wash for approximately 2 minutes.
This procedure ensures specific binding of the secondary antibody to its primary antibody, enabling accurate visualization in subsequent microscopy steps. The cells are now prepared for optional DAPI staining or mounting.
