Science Gateway > Protocols > Cell Biology Protocols - Table of Contents
Star Republic: Guide for Biologists

Staining for immunohistochemistry or FACS

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

  • PBT (PBS/0.1%Triton 100) is frequently used in place of PBS.
  • 0.5-5% BSA or goat/horse serum may be added as a blocking agent.