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Immunofluorescent Staining Protocol
Solutions and Reagents
- 1X Phosphate Buffered Saline (PBS): Dissolve 8g NaCl, 0.2g KCl, 1.15g Na2HPO4 and 0.2g KH2PO4 in 800mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature
- Poly-L-lysine solution: 0.1mg/ml in 1xPBS
- Glass coverslips No.1, 18mm dia.
- Fixation buffer: 4% paraformaldehyde in 1xPBS
- Permeabilization buffer : 0.1% Triton X-100 in 1xPBS
- Blocking buffer: 5% Fetal Bovine Serum (FBS) in 1xPBS
- Fluorescence-labeled secondary antibody
- Coat the coverslips with 0.1mg/ml poly-L-lysine solution at room temperature for 2hrs, dry, and then wash with 1xPBS buffer.
- Place the coated coverslip into each well of 12-well plate, and inoculate cells the day before immunocytochemistry experiment.
- Suck off the medium and rinse cells attached to cover slips twice with 1xPBS, removing liquid by gentle aspiration in this and subsequent steps.
- Fix cells with 4% paraformaldehyde in 1xPBS for 6 min at room temperature, and then rinse briefly twice with 1xPBS.
- The fixed cells can be permeabilize with 0.1% Triton X-100 in 1xPBS for 6 min.
- Wash cells briefly twice with 1xPBS, then block the coverslip with blocking buffer briefly at R/T.
- Dilute primary antibody with blocking buffer, and incubate the coverslip for 60 min at room temperature.
Note: You may wish to leave one slip for a secondary antibody only control.
- Wash cells 3 times with 1xPBS, then 2 times with blocking buffer.
- Incubate cells with a dilution of the fluorescence-labeled secondary antibody in blocking buffer for 30–45 minutes at room temperature in the dark.
- Wash cells three times with 1xPBS.
- Mount the coverslip on a glass slide. Store the slides in the dark.