Samples were then washed three times in 2x SSC + 0.1% Tween 20 for 20 minutes per wash and three times in 0.2x SSC + 0.1% Tween 20 for 30 minutes per wash (all at 60°C) before being washed twice in maleic acid buffer (MAB) for 15 minutes per wash at room temperature.
The tissue was washed 3 times in PBS for 10 minutes per wash and cryoprotected in a 30% sucrose solution in PBS overnight at 4°C.
Slides were then washed seven times in TNT buffer for 3 minutes per wash, once in PBS containing 1% Triton X to minimize background fluorescence (3 minutes) and a final wash in TNT buffer (3 minutes).
Following the Xylene baths, the slides were then rehydrated in successive decreasing concentration alcohol baths, from 100% to 95% to 70%, all for 5 minutes and then finally washed three times in PBS 0.1 M for 5 minutes per wash.
Slides containing these sections were processed to reveal Nissl substance beginning with a brief fixation in 4% paraformaldehyde in 0.1M PB for 15 minutes after which they were washed in distilled water three times for 2 minutes per wash.
Subsequently, the sections were washed seven times in TRIS-NaCl-Tween 20 (TNT) buffer solution for 3 minutes per wash followed by incubation with the secondary antibodies for 30 minutes at room temperature.
Node stage embryos were dissected in ice-cold PBS and fixed in the ice-cold 4% paraformaldehyde in PBS, pH 7.4 for 2 hours at 4 C. Embryos were washed 4 times, 30 minutes per wash, in PBS at 4C and then incubated in BLOCK (PBS +0.1% TX100 +2%BSA) for 1 hour at room temperature.
After being rinsed twice with PBS-T for fifteen minutes per wash, the blots were incubated at room temperature for one hour with primary antibody (HA, 1∶5000, from Covance, Skp1 1∶500, from Santa Cruz Biotech) in 0.5% non-fat milk in PBS-T and then rinsed again, twice with PBS-T.
