Immunochemistry Protocols: Immunofluorescence

Step 1

Rinse cells attached to cover slips twice with PBS, removing liquid by gentle aspiration in this and subsequent steps.

Step 2

Fixation permeabilization:

Option I:

• Fix cells with 4% formaldehyde in PBS for 6 min at room temperature, then rinse briefly twice with PBS**.
• Permeabilize fixed cells with 0.2% Triton X-100 in PBS for 6 min.

Option II:

• Fix/permeabilize cells in -20C methanol for 6 minutes**.

[**Note: At this stage, the procedure may be interrupted for long term storage. Coverslips immersed in PBS and stored at 4C are good for several weeks.]

Step 3

Wash cells briefly 3 times with PBS, then 2 times with PBS containing 5% BSA (blocking reagent).

Step 4

Dilute primary antibody in PBS/5% BSA. Working quickly, aspirate area surrounding coverslip to dryness, then gently add 100 ml of diluted primary antibody to the coverslip, so that solution remains restricted to coverslip by surface tension. Incubate for 1 hour at room temperature in a moist environment to prevent drying.

Step 5

Wash cells 3 times with PBS, then 2 times with PBS/5% BSA.

Step 6

Dilute fluorochrome-coupled secondary antibody in PBS/5% BSA and apply as in step 5. Incubate 1 hr. at room temperature.

Step 7

Wash cells 3 times with PBS, then mount coverslips to slides using antifade mounting medium.

Considerations: