LIFE01200604005 Shri Somnath Ghosh - Homi Bhabha National ...

CHAPTER 2
MATERIALS AND METHODS
PBS. Antibodies were diluted (1:200) in 1% BSA in PBS. Cells were incubated with primary
antibodies for 1 h 30 min at room temperature, washed three times in PBS and incubated with
secondary antibodies for 1 h at room temperature. Finally, cells were rinsed and mounted with
ProLong Gold antifede with DAPI mounting media (Molecular Probe, USA). Images were
captured using Carl Zeiss confocal microscope. Acquisition settings were optimized to obtain
maximal signal in immunostained cells with minimal background.
Table 2.6: The sequences of gene specific primers and their annealing temperature (Tm) used in
RT-PCR experiments
Gene Primer Sequence Tm ( o C)
β-Actin
F 5’-TGGAATCCTGTGGCATCCATGAAAC-3’
R 5’- TAAAACGCAGCTCAGTAACAGTCCG-3’
55
iNOS F 5’- CTCTGACAGCCCAGAGTTCC - 3’
62
R 5’- AGGCAAAGGAGGAGAAGGAG- 3’
p21 F 5’- GCCTTAGCCCTCACTCTGTG- 3’
58
R 5’-GGTTGGGAGGGGCTTAAATA- 3’
p53 F 5’- CGGGTGGAAGGAAATTTGTA- 3’
58
R 5’- CTTCTGTACGGCGGTCTCTC - 3’
p65 F 5’- ACAACTGAGCCCATGCTGAT- 3’
50
ATM
ATR
R 5’- GAGAGGTCCATGTCCGCAAT- 3’
F 5’-GGACAGTGGAGGCACAAAAT-3’
R 5’-GTGTCGAAGACAGCTGGTGA-3’
F 5’-CTCGCTGAACTGTACGTGGA-3’
R 5’-GCATAGCTCGACCATGGATT-3’
57
57
DNA-PK F 5’-TGCCAATCCAGCAGTCATTA-3’ 64
87