Immunofluorescence Protocol for Cell-based Assays Requiring Methanol Fixation

IMPORTANT: This protocol is recommended for both unconjugated and fluorophore conjugated antibodies. Please refer to the Product Usage Information section on the product webpage or product datasheet to determine if this product is validated and approved for use on cultured cell lines (IF-IC).

NOTE: Some CST™ antibodies work optimally using an alternate protocol. Please see the protocol on the product webpage for product-specific recommendations.

A. Solutions and Reagents

Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our Immunofluorescence Application Solutions Kit (#12727).

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade 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. Methanol: (#13604) 100%, Use ice-cold.
3. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer (#12411), or prepare a 1X PBS / 5% normal serum / 0.3% Triton™ X-100 buffer by adding 0.5 mL normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) and 30 µL Triton™ X-100 to 9.5 mL 1X PBS. Store at 4°C.
4. 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 0.1 g BSA (#9998) and 30 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 ice-cold 100% methanol.
2. Allow cells to fix for 15 min on ice or at 4°C.
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.

NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.

6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours 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.