Alexa488 coupled antibodies

Bovine and murine COCs or CC after in vitro culture were fixed during 30 min in 4% paraformaldehyde solution in PBS (pH 7.4). After fixation, cell permeabilization was performed in PBS/BSA 0.5%/Triton X100 0.5% for 15 min. Cells were then incubated in PBS/BSA 2% supplemented with 5% goat or horse serum for 1 h to block non-specific binding sites. A wash with PBS/BSA 0.1%/Tween 20 0.1% preceded the overnight incubation with the primary antibodies at 4°C. Goat antibodies against recombinant human (rh) TRIB1 (Santa Cruz Biotechnologies, Heidelberg, Germany), rabbit rh TRIB2 antibody and rabbit rh TRIB3 antibody (Sigma) were used at 1:100 dilution. Four 15-min washes preceded the incubation of the secondary donkey anti-goat or goat anti-rabbit Alexa488-coupled antibodies (Molecular Probes, Invitrogen) at room temperature for 2 h. Four PBS/BSA 0.1%/Tween 0.1% washes of 20 min were performed and 1 μg/mL of Hoechst was added in the last one. Lastly a drop of Mowiol® anti-fade solution was added to mount the slides and allow confocal microscopy analysis. Immunofluorescence was observed using a Zeiss confocal microscope LSM700 (Carl Zeiss Microscopy GmbH, Munich, Germany) using oil 40× and 63× objectives.