Immunofluoresence

Reagents

• Fixative: Neutral buffered formalin or 4% Paraformaldehyde in PBS or ice cold 10% methanol
• Cold PBS
• 100 mM Glycine in PBS
• 0.1% Triton X-100 in PBS. Can use other permeabilization agents if required
• Blocking Solution: 1% BSA
• Vectashield

Protocol

1. Prepare Cells at required density 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
6. Wash once with PBS
7. Permeabilize for 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
10. Incubate overnight with primary antibody in 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
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.

• It is possible to use ImageJ to analyse Colocalization