C2 confocal scanner

Cells were cultured on glass coverslips for at least 24 h before cell cycle synchronization. For plasmid-borne protein expression, synchronization was initiated 24 h post-transfection. Cells at indicated time points in G1 and S phase were washed with PBS and extracted in ice cold extraction buffer (10 mM PIPES, pH 6.8, 300 mM sucrose, 100 mM NaCl, 1 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 0.05% Triton X-100 and protease inhibitor cocktail). Cells were fixed using 4% paraformaldehyde for 15 min and permeabilized in 0.25% Triton-X-100 in PBS for 15 min at RT. After blocking in 1% BSA in PBS + 0.1% Tween-20 for 30 min, cells were incubated overnight at 4°C with primary antibodies: anti-BLM (C-18) (SCBT), anti-MCM6 (H-8) (SCBT), anti-Mcm2 (E-8) (SCBT), anti-Mcm7 (141.2) (SCBT), anti-Cdt1 (EPR17891) (Abcam), anti-53BP1 (E-10) (SCBT) or anti-PML (PG-M3) (SCBT) followed by incubation with Alexa Fluor labeled secondary antibodies. Specificity of BLM immunostaining with anti-BLM (SCBT) and anti-BLM (Abcam) was verified in BLM^KO cells. Images were acquired with a PerkinElmer UltraVIEW ERS spinning disc confocal imager equipped with a 63×/1.4 Oil DIC Plan-Apochromat objective, or with a Nikon C2 Confocal Scanner with a 60×/1.4 Oil DIC CFI Plan Apochromat Lambda objective.

Confocal microscopy of alizarin red‐stained larval teeth was performed using an inverted microscope (Ti; Nikon) with a C2+ confocal scanner (Nikon) and the following scanner settings: first filter cube, 447/60; second filter cube, 525/50561 LP; emission wavelength, 785 nm; laser power, 3.4; and pinhole size, 30 μm².