The nonspecific interactions were saturated with PBS containing 0.1% BSA and 0.01% Tween-20 (PBT buffer) for an additional overnight at 4°C.
Antibody coating was carried out by placing the formvar coated side of the grid faces down on a drop of carbonate-bicarbonate buffer with capture antibody for an overnight period at 4°C.
Next, the slide was returned to the dissecting scope and the spermathecae collected and transferred to a 1.5 ml centrifuge in 500 ml of lysis buffer for overnight incubation and subsequent DNA extraction and sperm number quantification as described in the previous sections.
Precipitate was collected by centrifugation at 8000 rpm for 20 min at 4 °C and dissolved in 50 mM Na-phosphate buffer having pH 7.4 and dialysed against the same buffer for overnight.
For cloning of methylated DNA fragments, the band of highest molecular weight in the HpaII lane was excised from a 1.0% low melting agarose gel, heated 10 min at 70°C and supplemented immediately with Bsp143I and restriction buffer for overnight digestion.
The sperm bundles were transferred to 500 ml of lysis buffer for overnight incubation and subsequent DNA extraction and sperm number quantification as described in the previous section.
Wild-type or mutant larvae were fixed with 4% paraformaldehyde and 1% glutaraldehyde, 1% paraformaldehyde in 100 mM cacodylate buffer for 1 h each at room temperature, followed by further fixation with 2% paraformaldehyde and 2% glutaraldehyde in 100 mM cacodylate buffer for 1 day and an overnight wash in 100 mM cacodylate buffer at 4°C.
Samples were pre-fixed with 2% PFA and 2% glutaraldehyde (GA, in 30 mM HEPES buffer (pH7.4)) for overnight at 4°C, followed by post-fixation with aldehyde OsO4 mixture (1.25% GA, 1% PFA, 0.32% K3[Fe CN 6], and 1% OsO4 in 30 mM HEPES buffer (pH 7.4)) for 2 hr at room temperature as previously described (Shirato et al., 2006).
The membrane was then blocked in blocking buffer containing 5% (w/v) nonfat dry milk and 1% (v/v) Tween 20 (T) in 20 mM trisbuffered saline (TBS, pH 7.6) for 1 h at 37°C, followed by incubation with the appropriate monoclonal or polyclonal primary antibody in blocking buffer for 1 h to overnight at 4°C.
In in vitro binding assay, recombinant GST-tagged PUM2 (40 µg) on Glutathione-Sepharose beads was incubated with 100 µg purified recombinant His-tagged Aurora-A in binding buffer at 4°C for overnight.
