Note: This method is suitable for immunofluorescence on cultured cells.
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.
6. Add 200µl blocking buffer per well to the chamber sections and incubate 60 min at room temperature.
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.