Immunofluorescence
1. Cell growth and feeding for IF
A. Seed cells in 4-chamber slides at 20,000 per chamber.
B. Grow to medium confluence
C. Feed with MCDB170+IP at -48 and -24 hr.
2. Fixing cells for IF
A. Wash cells (~70-80% confluent) with 1XPBS
B. Fix slides each in 1:1 ice cold MEOH:acetone and place at -20C for 10 minutes.
C. Store no more than 48 hr in 100% ethanol.
3. IF for hTERT
A. Remove fixative/ethanol from slides.
B. Add 1 ml 2N HCl to each chamber.
C. Incubate for 20 minutes.
D. Remove the HCl and neutralize with 1 ml 0.1 M Na-borate.
E. Incubate for 5 minutes.
F. Remove Na-borate and add 1 ml blocking buffer.
G. Incubate for 2 hr at RT.
H. Prepare NB 100-297 at indicated dilution.
I. Incubate ON at 4C.
J. Wash 4X5 min. in RT PBS.
K. Add secondary (FITC conjugated rabbit anti-mouse IgM).
L. Incubate at RT for 2 hrs.
M. Wash 4X5 min. in 1X PBS.
N. Wash 5 min in 1X PBS with DAPI (1.5 ug/ml).
O. Rinse slides briefly on PBS.
P. Remove chambers from slides.
Q. Mount in Vectashield (Vector catalog # H1200) and observe.
Blocking buffer To 500 ml of 1X PBS:
A. 5 g fish gelatin (Sigma catalog #G7765)
B. 25 ml goat serum
C. 5 g BSA Filter through 0.2 u filter and store at 4C
