善本生物

Methanol Method

Note: In this protocol, we use poly-D-lysine-coated glass coverslips to grow cells in a 12-well plate. The volume of the solution in this protocol can be adjusted to match the surface area of the wells or chamber slides that you use in your experiment.    

I. Preparations:

• Fixative solution: 4% Paraformaldehyde in 1x PBS
• Permeabilization solution: 100% Methanol
• Blocking solution: 5% Normal serum and 0.3% Triton X-100 in 1x PBS
• Coverslip coating. Coat the coverslips or chamber slides with 10 µg/ml poly-D-lysine in 1x PBS at 37℃ overnight, dry in a biosafety cabinet 
• Growing cell: Grow cells on coated coverslips or chamber slides till cells reach 70-80% confluence. Remove the culture medium, briefly wash the cells with 500 µL of 1x PBS twice before fixation

II. Cell fixation, permeabilization, and blocking:

1. Discard the PBS. Add 300 µL of ice-cold methanol, and incubate at–20℃for 10 minutes .

2. Carefully discard the methanol to a biohazard container and wash the cells with 500 µL of 1x PBS 3x 5 min.

3. Permeabilize the cells with 300 µL of the permeabilization solution at room temperature for 10 min.

4. Discard the solution and wash the cells with 500 µL of 1x PBS 3x 5 min.

5. Block the cells with 300 μL of the blocking solution at room temperature for 1h.

III. Immunostaining:

6. Dilute the primary antibody at the recommended dilution rate into the blocking buffer and add 200 μL of diluted primary solution to the cells.

7. Incubate the cells with the primary antibody  4℃ over night.

8. Discard the primary antibody solution, and wash the cells with 500 µL of 1x PBS 3x 5 min.

9. Dilute the fluorochrome-labeled secondary antibody at the recommended dilution rate into the blocking buffer, and add 200 μL of the diluted secondary antibody solution to the cells. A final concentration of 0.1 μg/mL DAPI can be added to the solution to stain the nuclei.

10. Incubate the cells with the secondary antibody at room temperature for 2h. Cover the plate with a aluminum foil to protect it from light.

11. Discard the secondary antibody solution, and wash the cells with 500 µL of 1x PBS 3x 5 min.

12. After the last wash, carefully remove the coverslips from the bottom of the well with forceps. Add one drop of anti-fade fluorescence mounting medium to the cells, and mount the cells onto a glass slide by flipping the coverslip over so the coverslip is face down.   

13. Seal the coverslip with nail polish by first dotting 4 drops around the coverslip, then connecting the dots with more nail polish to completely seal it .

14. Take images immediately using a regular fluorescence microscope or a laser scanning confocal microscope. For long-term storage, the slides should be wrapped in foil and stored in -20℃.