Following hybridization, the animals were taken through a series of seven washes, each lasting 1 hour at 55°C (100% HS/tRNA; 100%, HS/2 × SSC; 75% HS/2 × SSC, 50% HS/2 × SSC; 25% HS/2 × SSC; 2 × SSC/1% CHAPS, 2 × SSC/1% CHAPS).
After denaturation of chromosomal DNA in 70% formamide/2× SSC at 70°C, spreads were incubated in 2× SSC for 4 min at 70°C.
2× SSC was used to stop the reaction.
Next, slides were subsequently washed in 6× SSC, 2× SSC with 0.2 fi SDS, 2× SSC, and 1× SSC.
The slides were then washed in 2× SSC for 5 minutes and then equilibrated in 50% formamide/2× SSC for at least 15 minutes prior to denaturation.
Following hybridization, microarrays were washed in a series of four solutions containing 400 ml of 2× SSC with 0.05% SDS, 2× SSC, 1× SSC, and 0.2× SSC, respectively.
Membranes were washed at room temperature twice in 0.1% SDS with 2× SSC.
After washing with 2× SSC, cells were washed with PBS and mounted in Vectashield with DAPI.
Slides were then washed with 2× SSC and 0.1% SDS in 2× SSC at 50°C, 5 min each.
Slides were then rinsed in 2× SSC and incubated for 10 min in 50% formamide/2× SSC.
Slides were incubated overnight at 37°C, then washed in 50%formamide/50%% 2× SSC thrice for 5 min, 2× SSC for 5 min and 2× SSC/0.1% IGEPAL for 5 min, all at 46°C.
