Immunofluoresence

Reagents

• Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for cytoskeleton bound proteins) in PBS or ice cold 10% methanol (for lipid/membrane bound proteins)
• Cold PBS
• 100 mM Glycine in PBS
• 0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
• Blocking Solution: 2% BSA PBS
• Vectashield

Protocol

1. Prepare Cells at required density (quite sparse) in 12 well dishes on ethanol sterilized glass coverslips
2. Treat cells as required
3. Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
4. Wash twice with PBS
5. Add 200 uL of 100mM Glycine in PBS for 5 min to quench (up to 1 hour)
6. Wash once with PBS
7. Permeabilize for exactly 5 min with Triton X-100 (0.1% in PBS)
8. Wash three times with PBS (5 min each)
9. Block for 1-2h with 200 uL of blocking solution (PBS 2% BSA)
10. Incubate overnight with primary antibody in 200 ul blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
11. Wash coverslips 3 times 5 minutes with PBS with gentle rocking
12. Incubate in 500X secondary solution 45 minutes in 2% BSA PBS
13. Wash coverslips 3 times 10 minutes with PBS with gentle rocking
14. Add 10 uL vectashield to glass slide and place cells on slide. Fix with nail polish, dry for at least 1 hour.

• It is possible to use ImageJ to analyse Colocalization