Protocols | Morganis

IMMUNOFLUORESCENCE PROTOCOLS

1. Aspirate culture medium and briefly wash cells in PBS

2. Fix cells in 4% paraformaldehyde, 15 mins

3. Wash cells in PBS

4. Permeabilize cells in PBS + 0.1% Triton-X (PBST), 15 mins

5. Block cells in 1% bovine serum albumin (BSA) and 3% serum (from the host of chosen secondary antibody) in PBST, 30 mins

6. Incubate at 4°C with primary antibody diluted in PBST, overnight

7. Wash 3x 10 mins in PBST

8. Incubate with secondary antibody diluted in PBST, 2 hrs

9. Wash 3x 10 mins in PBST. Add nuclear stain (e.g. Hoechst/DAPI) to final wash if desired

10. Replace with PBS and image

For embryos from E5.0 - E9.0

1. Dissect embryos and briefly wash in PBS

2. Fix in 4% paraformaldehyde: E5.0-E7.5, 15 mins; >E7.5, 30 mins

3. Wash in PBS

4. Permeabilize in PBS + 0.5% Triton-X, 15 mins

5. Block at 4°C overnight in 1% BSA and 5% serum (from the host of chosen secondary antibody) in PBST (0.1% Triton-X)

6. Incubate overnight at 4°C with primary antibody diluted in PBST

7. Wash 3x 10 mins in PBST

8. Block again for 4 or more hours

8. Incubate overnight at 4°C with secondary antibody diluted in PBST. Add nuclear stain if desired

9. Wash 3x 10 mins in PBST

10. Replace with PBST and image

* All steps are carried out at room temperature unless otherwise specified

* Use PBS containing calcium and magnesium before fixation to preserve cell morphology

* Use PBST or PBS + BSA at all times when transferring embryos to prevent them from sticking to pipette tips