H 1000 10

HeLa cells were grown on glass cover slips (Corning Cat No. 2845–22). Upon processing for IF, cells were washed in PBS, pre-extracted (in 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM PIPES pH 7.0, 0.1% Triton X-100), washed with PBS, then fixed with 4% paraformaldehyde in PBS for 10 min at RT. The cells were then washed again with PBS and permeabilized with 0.1% Triton X-100 for 10 min at RT. Following this step, cells were washed with PBS and incubated with blocking buffer (0.5% BSA, 0.2% fish gelatin) for 1 h at RT. The cells were then incubated with FBL antibody diluted 1:250 in blocking buffer overnight in a humidified chamber at 4°C. The following day, cells were washed with PBS and incubated with secondary antibody (Abcam ab6564) diluted in blocking buffer for 45 min in a humidified chamber at RT. The cells were then washed in PBS and subjected to a final wash in 2× SSC with DAPI (Sigma-Aldrich D9542) for 10 min, followed by Vectashield (Vector Laboratories H-1000–10) treatment before mounting slides and imaging with a Zeiss LSM 710 confocal microscope. Images were visualized with FIJI Image software package.