Immunofluorescence (Immunocytochemistry)

IMPORTANT: This protocol employs an atypical fixation and denaturation 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. Ethanol , 70% solution, deionized.
3. 1.5 M Hydrochloric acid.
4. 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.
5. 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.
6. 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 and Permeabilization

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

1. Aspirate media then cover specimen with ice-cold 70% ethanol (use enough to cover completely to a depth of 3 - 5 mm, DO NOT LET DRY).
2. Allow to fix for 5 min.
3. Rinse three times in 1X PBS for 5 min each.
4. Aspirate PBS then incubate fixed specimen in 1.5 M HCl for 1 hour.
5. Rinse two times in 1X PBS for 5 min each.

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 specimen in an appropriate antifade reagent such as Prolong^® Gold Antifade Reagent (#9071) or Prolong^® Gold AntiFade Reagent with DAPI (#8961).
10. For long-term storage, store samples at 4°C protected from light.