Changes of washing solutions and preservation solution were much different from the original procedure of ACP 215 approved by the FDA.
The gels were washed in 2.5% Triton X-100 for 30 min with one change of wash buffer, incubated at 37°C for 60 72 hours in incubation buffer (50 mM Tris-HCl buffer pH 8, 5 mM CaCl2, 0.02% NaN3), stained with 5% Coomassie blue, and destained in 10% acetic acid and 40% methanol until proteinase bands were clearly visible.
22 Frequencies of key indicators from the structured interviews with pupils were used to determine reported changes of hand washing conditions over time, and Student t test comparisons were used to test the school-aggregated difference in these indicators between baseline and final data collection rounds for the intervention arms compared with the control arm.
The membrane was washed in two changes of low stringent wash buffer (2x SSC, 0.1 % SDS) for 5mins each at RT, and twice in high stringent wash buffers (0.5x SSC, 0.1 % SDS) for 15 min each at 65 °C.
After incubation, wash cells by centrifugation in 2-3 changes of sucrose-buffer wash consisting of 0.06 M Tris-Maleate buffer at pH 5.2 plus 10% sucrose.
with a minor change of replacing Wash 2 solution with 70% ethanol.
They were then spun down, removed of their supernatant, and resuspended and washed with several changes of PBS, then washed over night in 5 mL PBS.
Fixed buds were rinsed in five changes of 1 min washes in acetic buffer (10 mM sodium acetic, pH 4.5).
After blocking, the membranes were briefly rinsed in two changes of TBST, and washed once for 15 minutes and twice for 5 minutes in fresh changes of TBST.
The primary antibody was gently washed off with three changes of PBS and one wash with PBS/0.1% BSA, before the addition of 25 μl per gel of secondary antibody (5 μg ml−1 sheep anti-mouse IgG, fluorescein conjugated: Bioscience, Oxford, UK), which was incubated and washed off as before.
