Western blot analysis of HSP70 and OVGP1 proteins in BOECs

Total extracts of BOECs cultured (variant I) or co-cultured with embryos (variant II) at control (38.5°C) and elevated (41°C) temperatures were isolated. Thirty micrograms of BOEC total extracts (for HSP70 and OVGP1 detection) were resolved on 9% (for HSP70 detection) or 6% (for OVGP1 detection) SDS-polyacrylamide gels and transferred to Hybond-ECL nitrocellulose membranes. The membranes were initially blocked by gentle agitation in Tween 20-Tris-buffered saline containing 5% fat-free dry milk at room temperature for 1 h followed by overnight incubation at 4°C with mouse monoclonal antibody raised against HSP70 (concentration 1:2000). Monoclonal Anti-Heat Shock Protein 70 antibody (mouse IgG1 isotype) is derived from the BRM-22 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from BALB/c mice immunized with purified native full length bovine brain HSP70. For OVGP1 detection the rabbit polyclonal antibody raised against OVGP1 (concentration 1:1000) was used. An 18 amino acid synthetic peptide from near the N terminus of human OVGP1 ({"type":"entrez-protein","attrs":{"text":"NP_002548","term_id":"58386720","term_text":"NP_002548"}}NP_002548) has been used as an immunogen. Membranes were then washed and incubated with peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (concentration 1:20000) for 1 hat room temperature. Immunoreactive bands were detected using the enhanced chemiluminescence Western blot detection ECL Plus system (Pierce^™ ECL Plus Western Blotting Substrate, Thermo Fisher Scientific).

The expression of HSP70 and OVGP1 proteins in BOECs cultured alone (variant I) or co-cultured with embryos (variant II) at control and elevated temperatures (n = 3 for each of the 4 groups) was evaluated based on densitometric analysis using Biorad Molecular Imager FX and Quantity One software.

BOECs cultured alone (variant I) or co-cultured with embryos (variant II) at control (38.5°C) and elevated (41°C) temperatures were washed twice in PBS. Then, BOECs from each condition and temperature were fixed for 20 min in 4% (v/v) formaldehyde. After fixation and permeabilization in 0.5% Triton X(PBS-X) for 20 min, BOECs were washed twice for 10 min in PBS, blocked for 1 h at room temperature with 5% normal goat serum, and incubated with a mouse monoclonal antibody raised against HSP70 (concentration 1:100), or rabbit polyclonal antibody raised against OVGP1 (concentration 1:100) at 4°C for 18 h. Next, samples were washed and incubated with rabbit anti-mouse or goat anti-rabbit Alexa Fluor 488-conjugated secondary antibodies, respectively, (concentration 1:1000) for 1h at room temperature. Nuclei were counterstained with Hoechst 33342. Samples used as negative controls were stained only with secondary antibodies conjugated with Alexa Fluor 488 dye and counterstained with Hoechst 33342. All slides were mounted with Fluoromount (Sigma Aldrich F4680, USA). Analyses were carried out under a IX70 FV 500 confocal microscope (Olympus).

The immunofluorescence localizations of HSP70 and OVGP1 proteins in both BOECs variants at control (n = 5) and elevated (n = 5) temperatures and BOECs co-cultured with embryos at control (n = 5) and elevated (n = 5) temperatures were evaluated based on densitometry analysis using MicroImage software Olympus Optical CO. (Europa) GMBH.