1. Plate 400ml of a 105 cells/ml suspension into 8-chamber slides (plastic). Allow the cells to settle and attach for a couple hours.

2. Gently aspirate off the media and rinse cells 2-3X in PBS.

3. Rinse 1X in 100mM NaHPO4, pH 7.2-7.4 (diluted from 1M stock).

4. Fix 15-30' in 2.5% paraformaldehyde-NaHPO4.

5. Wash 5X in PBS (10' total).

6. Permeabilize cells in 0.25% saponin-PBS for 10' @ RT.

7. Quench cells in 100mM Glycine/10mM NH4Cl in PBS for 30'.

8. Block 30' in BPBS.

9. Incubate in primary antibody diluted in BPBS for 1-4h @ RT.

10. Wash 1X 15' in BPBS; 4X in PBS.

11. Incubate in secondary antibody:
a) fluorescence: 45'-2h in the dark; wash 3-5X in PBS (1-20' total); mount in Citifluor.
b)Vectastain: use biotinylated secondary; DAB-peroxidase substrate; mount in glycerol.

Fix 5ml:
2.5% paraformaldehyde 0.78ml 16% stock
100mM NaHPO4 0.5ml 1M NaHPO4, pH 7.2-7.4
3.82ml ddH2O

10X PBS
1.3M NaCl 76.0 g NaCl
0.07M Na2HPO4 9.94g Na2HPO4 (MW=142) H2O to 1L
0.03M NaH2PO4 3.6 g NaH2PO4 (MW=120)

Saponin-PBS 5ml:
0.25% saponin 0.125ml 10% stock
1X PBS 0.5ml 10X PBS
4.375ml ddH2O

10X Gly/NH4Cl 100ml:
1M Glycine (75.07) 7.5g
0.1M NH4Cl 0.535g H2O to 100ml

BPBS 10ml:
5% NGS 0.5ml Normal goat serum (or appropriate serum)
1.5% BSA 1.5ml 10%
0.05% Saponin 0.05ml 10%
1X PBS 1.0ml 10X