Image capture software

SDS-PAGE was done by using Cleaver Scientific (Rugby, UK) omniPAGE system. Proteins were transferred onto Millipore 0.45 μm nitrocellulose membrane. Immunoblotting was performed using TBS Tween (0.1%), containing 5% non-fat dry milk for blocking membrane and 1% non-fat dry milk for antibody solutions. Loading was controlled by developing membranes for β-actin or GAPDH. The following antibodies were applied: Rabbit PolyAb Anti-PARPI (Proteintech^®, Rosemont, IL, USA, 13371-1-AP), Rabbit PolyAb Anti-RIPK1 (Proteintech^®, 17519-1-AP), Rabbit PolyAb Anti-RIPK3 (Proteintech^®, 17563-1-AP), Anti-P-c-Jun (Cell Signaling, Danvers, MA, USA, 9261S), Anti-c-Jun (Cell Signaling, 9165S), and Anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA, 6C5). Rabbit PolyAb Anti-ACTB (Proteintech^®, 20536-1-AP), antiHRP-conjugated secondary antibodies: HRP-Goat Anti-Rabbit IgG (Proteintech^®, 00001-2), HRP-linked Anti-Mouse IgG (Proteintech^®, 7076S). The bands were visualized using a chemiluminescence detection kit (Thermo Scientific™, 32,106) and VWR™ (Radnor, PA, USA) Imager Chemi Premium gel documentation system with VWR™ Image Capture Software (version: 1.6.1.0). For densitometry analysis, Western blot data were acquired using ImageJ software bundled with 64-bit Java 1.8.0_172.

The fresh mature and vitrified oocytes were solubilized in reducing sample buffer, boiled for 5 min, separated in 12% SDS-PAGE (30 oocytes per lane), and transferred onto nitrocellulose membranes. After blocking with 5% non-fat milk in 0.1% Tween 20 in PBS, the membranes were incubated with primary antibodies: anti-CD9, anti-CD81, anti-CD151, anti-CD82, and anti-CD63 overnight at 4 °C, followed by 1 h of incubation with anti-rabbit/mouse IgG-HRP conjugate. Detection of actin was used as a loading control. The antibody reaction was visualized using SuperSignal West Pico Chemiluminescent Substrate. The HRP chemiluminescence was monitored with VWR^® Imager CHEMI Premium detection systems and analyzed using the VWR^® Image Capture Software.
Densitometric analysis was performed using ImageJ software. The relative density of individual tetraspanins in fresh and vitrified mature oocytes was calculated as the ratio of the optical density of antibodies to tetraspanins and actin in the blot. Results are presented as the mean ± SEM of three replicates. The statistical significance of differences was evaluated by Student’s t test using the statistical software Sigma Plot 11.0.