Fitc and rhodamine red conjugated goat anti rabbit or anti mouse igg

For Hec1 staining, cells were permeabilized in PHEM buffer supplemented with 0.1% Triton X-100 and protease inhibitors for 1 min on ice, fixed with 3.7% paraformaldehyde in PHEM buffer for 5 min at room temperature, washed three times with PBS, and immunostained as appropriate (Hua et al., 2011 (link)). For all other fluorescence microscopy applications, cells grown on coverslips were fixed in −20°C methanol for 10 min, blocked with 10% goat serum at 37°C for 30 min, incubated at 37°C with the primary antibodies for 2 h, washed extensively and probed with the FITC- and Rhodamine red–conjugated goat anti–rabbit or anti–mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 30 min. Coverslips were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Fluorescence images of fixed cells shown in Figs. 1 (A–C and G), 2 (A and B), 6 D, 8 B, S1 (D–G), S2 (A–C and F), and S3 (A and B) were acquired in a DM Digital Microscope (DM5000; AF6000 E acquisition software; Leica) at room temperature, using a 63× HC Plan-Apochromat, NA 1.40 oil-immersion objective. Images shown in Figs. 4 (B–D) and 8 (D and G) were acquired in a laser confocal microscope (TCS SP8; LAS-AF acquisition software; Leica) at room temperature, using a 63× HC Plan-Apochromat, NA 1.40 oil-immersion objective. Images were processed using Photoshop CS5 (version 12.0; Adobe).