Immunofluorescence Protocol: Cultured Cell

Note: This method is suitable for immunofluorescence on cultured cells.

Reagents of Immunofluorescence Protocol

• PBS: To prepare 1 L 1X PBS: dissolve 8g of NaCl, 0.2g of KCl, 1.44g of Na₂HPO₄, 0.24g of KH₂PO₄ in 800ml distilled H₂O, then adjust pH to 7.4 with HCl and adjust volume to 1L with additional distilled H₂O.
• Fixation solution: 4% Paraformaldehyde, in PBS.
• Permeablization solution: 0.3% Triton X-100 in PBS. To prepare 100 mL: add 300 µl Triton X-100 to 100 mL PBS and mix. (Optional)
• Blocking buffer: To prepare 10 ml, add 0.5 ml normal serum in PBS and mix well. While stirring, add 30 µl Triton™ X-100.
  NOTE: Select serum that are from the same species as the secondary antibody.
• Antibody dilution buffer: To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 1X PBS. Mix well then add 0.1g BSA.
• Antifade Reagent

Steps of Immunofluorescence Protocol

Sample Preparation of Immunofluorescence Protocol:

1. Grow cultured cells on cover slips or chamber slide.
2. Move the culture media and rinse with warn PBS.
3. Cover cells to a depth of 2–3 mm with warm Fixation solution and fix for 15 min at room temperature.
4. Rinse three times in 1X PBS for 5 min each.
5. Add permeablization solution and incubate for 3~5 min at room temperature.

NOTE: This step is optimized for intracellular staining.

Blocking of Immunofluorescence Protocol:

6. Add 200µl blocking buffer per well to the chamber sections and incubate 60 min at room temperature.

Staining of Immunofluorescence Protocol:

7. Dilute the primary antibody to the recommended concentration on the datasheet in antibody dilution buffer.

8. Add 200µl per well to the chamber slides and incubate two to three hours on a shaker at room temperature or overnight at 4°C.

NOTE: If using primary antibodies directly conjugated with fluorochrome, the incubation should be performed in the dark, and then skip to step 11.

9. Rinse three times in 1X PBS for 5 min each.

10. Prepare fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer and incubate for 2~3 hours at room temperature in the dark.

11. Rinse three times in 1X PBS for 5 min each.

12. Coverslip with anti-fade mounting medium.

13. Store at 4°C protecting from light.