P36935

The immunofluorescent microscopy was performed using methods previously described.⁷⁷ (link) Briefly, 1 × 10⁵ ovarian cancer cells (OVCAR3, OVCAR4 and PEO1) and negative control cells (OSEC) were seeded on coverslips and incubated overnight. The cells on coverslips were fixed in 4% PFA and incubated overnight with IgG antibodies extracted from ovarian tumour tissues diluted 1:100 in PBS and 1% FCS. The coverslips were then incubated with 1:200 anti-human IgG (Invitrogen, A-11013) and mounted with DAPI (Invitrogen, P36935). The images were taken using confocal microscope (Leica SP5). The confocal images were analysed using Fiji.

Cells were seeded in 35 mm glass bottom dishes (MatTek P35G-1.5-14-C) and treated as needed. At the end of treatment, cells were washed with PBS, and pre-extracted for 1 min with extraction buffer (50 mM PIPES pH 6.9, 25 mM KCl, 3 mM EGTA, 1 mM MgSO₄, 0.5% Triton X-100), followed by fixation in 4% paraformaldehyde in PBS (Alfa Aesar J61899) for 20 min at 4°C. Cells were washed 2× with PBS, permeabilized with 0.5% Triton-X100 in PBS for 10 min at RT, washed 2× with PBS and blocked for 1 h at RT (1% BSA, 10% goat serum in PBS). Primary antibodies were incubated overnight at 4°C in 1% BSA in PBS. Cells were washed 3x/5min with PBS, and incubated with secondary antibodies (1:2000, Invitrogen goat anti-mouse or anti-rabbit IgG (H+L) Alexa Fluor 488) in 1% BSA in PBS for 1 h at RT. After washing with PBS and drying, coverslips were mounted with Prolong Gold Antifade with DAPI (Invitrogen P36935). At least 50 cells were analyzed per condition. Images were taken with a Nikon Eclipse Ti-E epifluorescence microscope equipped with a 100x objective (Plan Apo, 100× 1.45, oil), a Photometrics CoolSnapHQ2 camera and appropriate filters (DAPI, GFP, Texas Red and Cy5). The numbers of foci per cell were quantified using Nikon Elements Software. Experiments were repeated in triplicate.

Cultured cells were washed with phosphate-buffered saline with calcium and magnesium (PBS⁺) and fixed for 5 min in 3.7% formaldehyde, washed with PBS, fixed for 5 min with methanol/acetone at −20 °C, washed, and permeabilized twice for 7 min with 0.1% Triton-X100. Cell smears and cryostat section were fixed for 10 min with 3.7% formaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100. Blocking was performed using 10% goat serum in PBS for at least 5 min. Cells/sections were incubated for 1 h with the primary antibody in 10% goat serum in PBS, washed three times, and incubated for 30 min with secondary antibody in 10% goat serum in PBS. For peroxidase staining, cells were incubated for 30 min with Ultravision ONE HPR polymer (TL-125-PHJ, Thermo Scientific) instead of secondary antibody. Cells were then washed with PBS and incubated for 10 min with 3’3-diaminobenzidine and 1:1000 30% hydrogen peroxide. The overview of antibodies used is summarized in Supplementary Table 3. Nuclei were counterstained with DAPI (P36935, Invitrogen).

Cells seeded on coverslips for more than 24 h were fixed in 4% formaldehyde for 30 min at room temperature, then permeabilizated with 0.1% Triton X-100 for 30 min. After that, cells were treated with the primary antibody overnight at 4°C, followed by incubation with species-specific secondary antibody for two hours at room temperature. The coverslips were mounted using an antifade mounting solution containing 4,6-diamidino- 2-phenylindole (DAPI; P36935, Invitrogen) after three-time washing with phosphate-buffered saline (PBS). Actin was stained with Myo10 (Rabbit, HPA024223, Sigma-Aldrich) and Rhodamine Phalloidin (R415, Invitrogen) following the manufacturer's instructions. Confocal images were captured using ZEISS LSM 880 confocal microscope with Airyscan (Carl Zeiss, Germany) or Leica TCS SP8 confocal microscope (Leica, Switzerland), and fluorescence quantification was measured as described previously 9 (link). Co-localization analysis was performed via Leica Application Suite X (LAS X).