Immunofluoresence
From Bridges Lab Protocols
Reagents
- Fixative: Neutral buffered formalin or 4% Paraformaldehyde (for lipid/membrane bound proteins) in PBS or ice cold 10% methanol (for cytoskeleton 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
- Prepare Cells at required density (quite sparse) 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 (up to 1 hour)
- Wash once with PBS
- Permeabilize for exactly 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 (PBS 2% BSA)
- Incubate overnight with primary antibody in 200 ul 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 45 minutes in 2% BSA PBS
- Wash coverslips 3 times 10 minutes with PBS with gentle rocking
- Add 10 uL vectashield to glass slide and place cells on slide (coverslip side up, use sharp tweezers and tip, 2 coverslips on each slide). Label slide with date, cells and treatment + color code for antibodies. Fix with nail polish, dry for at least 1 hour up to ON in dark drawer. Store long term in sealed box at 4C.
- It is possible to use ImageJ to analyse Colocalization
