Dm5500b upright microscope

Microscopy was performed as described previously (Lisby et al. 2004 (link); Mine-Hattab and Rothstein 2012 (link)). Cells were pelleted from treated or untreated cultures and resuspended at higher density before being placed on a 1.4% agarose slab for visualization. Images were acquired on a Leica DM5500B upright microscope (Leica Microsystems) illuminated with a 100-W mercury arc lamp. High-efficiency filter cubes were used for fluorophore imaging (Chroma 41028, Chroma 31044v2, and Chroma 41002C for YFP, CFP, and RFP, respectively). Images were captured with a Hamamatsu Orca AG cooled digital CCD (charge-coupled device), and analysis of image data was performed with Volocity software (Perkin-Elmer). For mobility experiments, we captured 15 z-stacks spaced by 300 nm every 10 sec for 70 time points. Exposure times were as follows: 30 msec for differential interference contrast (DIC), 100 msec for YFP, 100 msec for RFP, 800 msec for Rad52-CFP, and 2 sec for Ddc1-CFP. CFP images were taken as part of a complete stack of all colors performed before time-lapse imaging began. For Rad51 and Rad52 focus experiments, we captured 21 z-stacks spaced as for mobility experiments. DIC exposure time was 30 msec, and the YFP and CFP exposure time was 800 msec.

For staining of E-cadherin protein, the cells were fixed in 4% paraformaldehyde/PBS for 20 min and permeabilized with 0.1% Triton X-100/PBS for 3 min at room temperature. Subsequently, cells were blocked in 2% goat serum plus 1% BSA (both from Thermo Fisher Scientific, Inc.) in PBS for 30 min at room temperature, incubated with primary antibody at 4°C overnight, and then incubated with secondary antibody conjugated to Alexa Fluor 488 (cat. no. A32731; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. The nuclei were counterstained with DAPI. The primary antibody used was anti-E-cadherin (1:200; cat. no. ab40772; Abcam). Images were acquired using a light Leica DM5500B upright microscope (Leica Microsystems GmbH) with a Retiga SRV Cooled CCD camera (Teledyne Photometrics) and ImagePro Plus software (version 6.0; Media Cybernetics, Inc.).

Cells grown on coverslips were washed with phosphate buffered saline (PBS) and fixed 4% paraformaldehyde for 30 minutes. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 minutes at room temperature with gentle mixing. Cells were washed twice with PBS and then incubated with 3% bovine serum albumin (BSA)/0.1% Tween in PBS for 60 minutes. The cells were then incubated with an anti-V5 antibody (Life Technologies) at 1:200 dilution in 3%BSA/0.1%Tween for one hour at room temperature. The samples were washed twice in PBS followed by incubation with the anti-mouse Alexa Fluor 488 at 1:200 for one hour (Life Technologies). Cells were washed twice and mounted in ProLong Antifade mounting buffer with DAPI (Life Technologies) and left to cure overnight. Images were captured at 40x magnification on a Leica DM5500B upright microscope (Leica Microsystems, GmbH) using ImagePro Plus software (MediaCybernetics).