Protocol for Intracellular Staining
1. Cells to be stained should be in 12-well tissue culture or Mattech
2. Wash 3 times with COLD PBS and apirate
3. Fixing: Add 2-3ml of 2% Paraformaldehyde in PBS to each well. Fix overnight in
fixation buffer at 4˚ C.
4. Wash 3x with COLD PBS and aspirate
5. Incubate cells with 100 ul block
6. For Rabbit antibodies, use: 200 ul human IgG (Kohn Faction II/III 6 mg/ml) for
10 minutes. (Assumes Abs will not react with human IgG)
7. For Murine antibodies, block with Mouse IgG in PBS/Facs buffer
8. Add 1.0 ml permeabilization buffer (0.1% Saponin in FACS buffer) for 30 minutes
9. Add Primary antibody 0.5 µg/coverslip, and incubate 30 minutes on ice. (1/100
dilution)
10. Wash 3 times with cold FACS buffer.
11. Make up Secondary Antibody (0.5-1.0 µg/well) or 1:40 dilution in 100 µl of
Permeabilization Buffer and add to cells. Incubate 1 hr. at 4˚.
12. Wash cells 3 times with FACS buffer
13. Mount on glass slides using SloFade and Store covered at 4˚ in the dark.
FACS Buffer:
Permeabilization
Buffer:
PBS
1.0% Bovine Serum Albumin (BSA)
0.1% Sodium Azide
FACS Buffer plus 0.1% Saponin
Saponin Stock 10% in FACS buffer
For permeabilization Buffer (0.1%) add 100 ul of the stock solution to 10ml of FACS
buffer
Sigma S 4521
Costar Plates 96 well polypro plates (800)-492-1110
#3365-plates
#3790-lids
Human IgG Chon fraction II/III
Sigma G-4386
Stock at 12 mg/ml (72mg in 6 ml)
Working solution: Dilute 1:1 in FACS buffer
Dilutes in HEPES-BSA