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
- Prepare Cells at required density in 12 well dishes on ethanol sterilized glass coverslips
- Treat cells as required
- Wash cells twice with 2 mL ice cold PBS then fix for 10 min at RT with desired fixative with gentle rocking
- Wash twice with PBS
- Add 200 uL of 100mM Glycine in PBS for 5 min to quench
- Wash once with PBS
- Permeabilize for 5 min with Triton X-100 (0.1% in PBS)
- Wash three times with PBS (5 min each)
- Block for 1-2h with 200 uL of blocking solution
- Incubate overnight with primary antibody in blocking solution at 4C. Dilution varies with the antibody, but typically start with 200x
- Wash coverslips 3 times 5 minutes with PBS with gentle rocking
- Incubate in 500X secondary solution
- Wash coverslips 3 times 10 minutes with PBS with gentle rocking
- 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