Immunofluorescence (Immunocytochemistry), Blocking Buffer-free Protocol with Formaldehyde Fixation

IMPORTANT: This protocol is designed for unconjugated primary antibodies that are sensitive to serum blocking. When applicable, it will be linked under the Product Information section on an antibody's product-specific webpage.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

1. 1X Phosphate Buffered Saline (PBS) : To prepare 1 L 1X PBS: add 100 ml 10X PBS ( #12528 ) to 900 ml water, mix. Adjust pH to 8.0.
2. 4% Formaldehyde, Methanol-Free ( #47746 ): Use fresh. Dilute concentrated stocks in 1X PBS.
3. Antibody Dilution Buffer : Purchase ready-to-use Immunofluorescence Antibody Dilution Buffer ( #12378 ), or prepare a 1X PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 30 µl Triton^™ X-100 and 0.1 g BSA to 10 ml 1X PBS. Mix well.
4. Fluorochrome-conjugated Secondary Antibody: Use a secondary antibody that is reactive to your primary antibody host species (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.
5. Mounting medium: Prolong ^® Gold AntiFade Reagent ( #9071 ) or Prolong ^® Gold AntiFade Reagent with DAPI ( #8961 ).

B. Fixation

1. Fix cells with 4% formaldehyde for 15 minutes at room temperature.
2. Aspirate fixative, wash three times in 1X PBS for 5 minutes each.
3. Proceed with Section C.

C. Immunostaining

NOTE: Do not allow cells to dry at any time during this procedure and protect sensitive fluorophores from light.

1. Aspirate 1X PBS and then permeabilize cells with Antibody Dilution Buffer for 60 minutes.
2. Prepare primary antibody in Antibody Dilution Buffer (see product website for recommended dilution range).
3. Aspirate Antibody Dilution Buffer, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Wash three times with 1X PBS for 5 minutes each.
6. Incubate cells in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in the dark.
7. Wash three times in 1X PBS for 5 minutes each.
8. Counterstain as appropriate and then mount samples for imaging.
9. For long term storage, store stained samples at 4°C protected from light.