Immunofluorescence on Human Embryonic Stem Cells

Main Materials:
Rabbit anti-Oct-4 antibodies from Santa Cruz
Mouse anti-SSEA-1, -3, -4,-Tra1-60, and -Tra1-81 from Chemicon

1. Grow stem cells on feeders in 12- or 24-well plate until ready
2. Remove media and fix with 4% paraformaldehyde for 20 min at Room Temp.
3. Wash with 0.1% Tween-20/PBS, 5 minutes each, 2X at RT
4. (Oct-4 only) Permeablize with 1% TritonX/PBS for 30 minutes at RT
5. Block with 4% Goat Serum/PBS, 30 minutes, RT
6. Add 1º antibodies/PBS at 1:200 dilution, overnight at 4ºC
7. Wash with 0.1% Tween-20/PBS, 5 minutes each, 3X at RT
8. Detect with conjugated 2º antibodies/PBS at 1:300 dilution, 1 hour at RT
9. Wash with 0.1% Tween-20/PBS, 5 minutes each, 3X at RT
10. Counter stain with DAPI/PBS (add 1 ul of 1mg/mL DAPI to 1 mL PBS)

5 Methyl Cytosine Immunostaining
Mouse anti-5meC from Genway
Adapted from Stantos et al.

Procedures

1. Wash cells in PBS
2. Fix 4% paraformaldehyde/PBS, 20 minutes
3. Permeablize with 1% Triton X-100, 30 min – 1 hour
4. Wash with 0.1% Tween-20/PBS, 5 min
5. Treat with 4 M HCl/0.1% Triton X-100, 10 minutes
6. Neutralize with 100 mM Tris/HCl (pH 8.5), 30 minutes
7. Block with 1% BSA/0.1% Tween-20/PBS at 4ºC, overnight
8. Stain with mouse monoclonal antibody to 5meC, diluted 1:200, 2-4 hrs. at RT
9. Wash with 0.1% Tween-20/PBS, 2 times
10. Detect with secondary antibody, 1:300 dilution, 1 hr at RT
11. Counterstain with DAPI