Pluripotency markers staining
Hanqin Li, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the standard procedure for staining pluripotency markers, e.g. OCT4, SSEA4, alkaline phosphatase, and etc. on human pluripotent stem cells (hPSCs).
Protocol overview
A. Immunofluorescence staining
B. Alkaline phosphatase staining
General notes
Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
Steps
A. Immunofluorescence staining
Wash cells once with PBS
Phosphate buffer saline, PBS, pH 7.4
| A | B |
|---|---|
| NaCl | 137 mM |
| KCl | 2.7 mM |
| Na2HPO4 | 10 mM |
| KH2PO4 | 1.8 mM |
| CaCl2•2H2O | 1 mM |
| MgCl2•6H2O | 0.5 mM |
Fix cells with 4% PFA at 4Room temperature for 0h 15m 0s
4% PFA, pH 7.4
| A | B |
|---|---|
| PBS | 500 ml |
| PFA | 20 g |
| NaOH | Adjust pH to 7.4 |
Final volume: 500 ml
Wash cells twice with PBS, incubate 0h 5m 0s in between washes at Room temperature
Incubate in PBST for 0h 30m 0s at Room temperature to permeabilize cell membrane
PBST, pH 7.4
| A | B |
|---|---|
| PBS | 1x |
| Triton-X100 | 0.3% |
Incubate in 3% Bovine Serum Albumin (BSA) for 1h 0m 0s at Room temperature
3% Bovine Serum Albumin (BSA)
| A | B |
|---|---|
| PBS | 500 ml |
| BSA | 15 g |
Incubate with primary antibody (1:200) in 3% BSA at 4°C 1h 0m 0s
Wash three times with PBS, incubate 0h 5m 0s in between washes at 4Room temperature
Incubate with secondary antibody (1:1,000) in 3% BSA at 4Room temperature for 1h 0m 0s in the dark and in a humidified chamber.
Wash once with PBS
Incubate with 0.1 µg/ml DAPI for 0h 5m 0s at 4Room temperature
Wash twice with PBS, incubate 0h 5m 0s in between washes at 4Room temperature
Image cells, seal the plate with parafilm, and wrap with foil for longer storage at 4°C. Florescence is usually stable for several weeks.
B. Alkaline phosphatase staining
Wash once with PBS
Fix cells with cold 4% PFA, 0h 10m 0s at 4Room temperature.
Wash twice with PBS
Incubate cells with 100 mM Tris, pH 9.5, 0h 10m 0s at 4Room temperature
100 mM Tris, pH 9.5
| A | B |
|---|---|
| Tris | 6.57g |
| HCl | Adjust pH to 9.5 |
Final volume: 500 ml
Add NBT/BCIP substrate in 100 mM Tris, and let reaction develop at 4Room temperature, avoiding light until color becomes clearly apparent in control cells.
Wash in PBS and keep in PBS (violet product is soluble in organic solvent).
