Immunostaining Protocol for Microtubule Visualization

Immunostaining was performed using similar procedures as described previously (Foe and von Dassow, 2008 (link); Minc et al., 2011 (link)). The fixation was performed in bulk (Figs. 2 A and S1 F), in the flat PDMS perfusion chamber (Figs. 2 C and S2 G), or in microwells (Fig. S2 J). Fixations in chamber or microwells were done under the microscope to ensure that eggs do not move or change shape during liquid exchange. All experiments involved similar protocols. Eggs were first fixed for 70 min in 100 mM Hepes, pH 6.9, 50 mM EGTA, 10 mM MgSO₄, 2% formaldehyde, 0.2% glutaraldehyde, 0.2% Triton X-100, and 400 mM glucose. Eggs were then rinsed three times for 10 min in PBS plus Tween 20 (PBT) and one time in PBS and placed in 0.1% NaBH₄ in PBS made fresh for 30 min. Eggs were rinsed again with PBS and PBT and blocked in PBT plus 5% goat serum and 0.1% BSA for 30 min. For MT staining, cells were incubated for 48 h with a primary anti–α-tubulin antibody, clone DM 1A (Sigma-Aldrich) at 1/8,000, rinsed twice in PBS, and then incubated for 4 h with fluorophore-conjugated anti-mouse secondary antibody (Sigma-Aldrich) at 1/750. Aster staining after laser ablation was done in a PDMS flow chamber, and the fixative was introduced 10 s after ablation.