Foci immunofluorescence

HeLa WT and cavin3 KO cells were pre-permeabilized with CSK buffer (10 mM HEPES, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 0.7% Triton X-100) for 5 min and were then fixed with 4% PFA/PBS for 15 min, permeabilized with 0.5% Triton X-100 solution for 15 min followed by blocking for 1 hr RT. Cells were then immunostained with primary antibodies against mouse BRCA1 alone (Santa Cruz, Cat# sc-6954, RRID:AB_626761, IF 1:50), Rap80 alone (Cell Signaling Technology, Cat# 14466, RRID:AB_2798487, IF 1: 50), γH2AX alone (Abcam, Cat# 20669, RRID:AB_445689, IF 1:100), and the appropriate Alexa Fluor 488 Goat anti-Rabbit IgG (H + L) (Thermo Fisher Scientific, Cat# A-11034, IF 1:500) conjugated secondary antibodies. Images were taken with a Zeiss microscope. Quantification of the percent of cells was based on foci formation (more than 5 foci/nucleus) was determined from more than 500 cells/experimental condition from 2 to 3 independent experiments using an automated plugin for ImageJ.