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Immunofluorescence Protocol for Cell-based Assays Requiring 2% Formaldehyde Fixation and 0.1% Triton™ X-100 Permeabilization

IMPORTANT: This protocol employs an atypical fixation and permeabilization strategy with which only certain targets are compatible. Where appropriate, this protocol will be linked to its validated antibody under the Product Information banner on the 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 1L 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline (#12528) to 900 mL dH2O, mix. Adjust pH to 8.0.
  2. 2% Formaldehyde, Methanol-Free: To prepare 10 mL, add 1.25 mL 16% Formaldehyde, Methanol-Free (#12606) to 8.75 mL 1X PBS and mix well. Prepare fresh just prior to use.
  3. Blocking Buffer (1X PBS / 5% normal serum / 0.1% Triton™ X-100): To prepare 10 mL, add 0.5 mL normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 10 µL Triton™ X-100 to 9.5 mL 1X PBS. Store at 4°C.
  4. Antibody Dilution Buffer (1X PBS / 0.1% Triton™ X-100): To prepare 10 mL, add 10 µL Triton™ X-100 to 10 mL 1X PBS. Store at 4°C.
  5. 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.

B. Fixation

NOTE: All subsequent incubations should be carried out at room temperature (20-25°C) unless noted otherwise.

  1. Aspirate culture media then cover cells to a depth of 2–3 mm with 2% formaldehyde.
  2. Allow cells to fix for 15 min.
  3. Rinse three times in 1X PBS for 5 min each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody in Antibody Dilution Buffer (see product website for recommended dilution range).
  3. Aspirate blocking solution then apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr protected from light.
  7. Rinse three times in 1X PBS for 5 min each protected from light.
  8. Counterstain as appropriate.

    NOTE: When including fluorescent cellular dyes in your experiment (DNA dyes, etc.), please refer to the dye product page for its recommended protocol. View our listing of cellular dyes validated for use in immunofluorescence.

  9. Mount samples for imaging.
  10. For long-term storage, store samples at 4°C protected from light.