Zen 2 pro

Cells were seeded on coverslips for 12–24 h, washed once in Dulbecco’s Phosphate Buffer Saline (DPBS, Lonza) containing Calcium and Manganese and fixed with 4% paraformaldehyde (PAF) in PBS (Phosphate Buffer Saline) pH 7.3, for 30 min at room temperature. Coverslips were subsequently washed three times with PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min then washed three times with PBS. Coverslips were then saturated for 1 h in blocking buffer [0.2% gelatin, 2% Bovine Serum Albumin (BSA), 2% FBS (Lonza) in PBS], followed by the incubation for 1 h with primary antibody diluted at 1:100 in blocking buffer, except for GPP130 that was diluted at 1:300. After washing with PBS, cells were incubated for 1 h with Alexa 488- or Alexa 568- secondary antibody (Life Technologies) diluted at 1:600 in blocking buffer. After three washes with PBS, nuclei were labeled with DAPI 1:300 for 15 min and then coverslips were mounted on glass slides with Mowiol. Fluorescence was detected through an inverted Zeiss LSM700 confocal microscope. Acquisitions were done with ZEN pro 2.1 software (Zeiss, Oberkochen, Germany).

Cells were seeded on coverslips for 12 to 24h, washed once in Dulbecco’s Phosphate Buffer Saline (DPBS, Lonza) and fixed either with 4% paraformaldehyde (PAF) in PBS pH 7.3, for 30 min at room temperature or with ice-cold methanol for 10 minutes at room temperature. Coverslips were then washed three times with PBS. Only if the fixation had been done with PAF, cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min then washed three times with PBS. Coverslips were then put in saturation for 1h in blocking buffer [0.2% gelatin, 2% Bovin Serum Albumin (BSA), 2% Fetal Bovine Serum (FBS) (Lonza) in PBS], followed by the incubation for 1h with primary antibody diluted at 1:100 in blocking buffer. After washing with PBS, cells were incubated for 1h with Alexa 488-, Alexa 568- or Alexa 700-conjugated secondary antibody (Life Technologies) diluted at 1:600 in blocking buffer. After three washing with PBS, coverslips were mounted on glass slides with Mowiol. Fluorescence was detected through an inverted Zeiss LSM780 confocal microscope. Acquisition were done using the ZEN pro 2.1 software (Zeiss, Oberkochen, Germany). For selective membrane permeabilization, we have used digitonine. Stock solution was prepared at 5mg/mL in absolute ethanol, 0,3M sucrose, 0,1M KCl, 2,5 mM MgCl2, 1 mM EDTA, 10 mM HEPES, pH 6,9. Permeabilization was done at 4°C during 15min.