1. Wash cells with PBS.

2. Fix cells. For immunohistochemistry, cells can be fixed on plate or slides. For FACS, cells must be detached in a single-cell suspension.

3. Wash cells three times with PBS.

4. Incubate with the primary antibody at the desired dilution (1:100 - 1: 10,000) in PBS. Incubate at room temperature for 30 minutes to 4 hours, or 4 °C for 2 hours to overnight with gentle rocking.

5. Wash three times with PBS for a total of at least 20 minutes with gentle rocking.

6. Incubate with the secondary antibody at the desired dilution (1:100 - 1: 1,000) in PBS. The secondary is against the primary antibody, e.g., if the primary antibody is rabbit IgG, then the secondary antibody could be mouse anti-rabbit-IgG or goat anti-rabbit-IgG. Incubate at room temperature for 30 minutes to 4 hours, or 4 °C for 2 hours to overnight with gentle rocking.

7. Wash three times with PBS for a total of at least 20 minutes with gentle rocking.

Fluorescein-labeled secondary antibody

8a. Analysis by flow cytometry or microscopy.

Biotin-labeled secondary antibody

8b. Incubate with the Streptavidin-Fluorescein in PBS. Incubate at room temperature for 30 minutes to 4 hours, or 4 °C for 2 hours to overnight with gentle rocking.

9b. Wash three times with PBS for a total of at least 20 minutes with gentle rocking.

10b. Analysis by flow cytometry or microscopy.

Note