Anti cx43

Tissues were fixed in 10% neutral buffered formalin overnight at room temperature. The fixed tissues were embedded in paraffin and cut into 4 to 6 μm tissue sections. Then, the slides were stained with hematoxylin–eosin (HE), anti-HIF-1α (Abcam), anti-Cx43 (Santa Cruz), anti-BCR-ABL (Abcam), anti-BAX (Boster), and anti-Ki67 (Bioss, Beijing, China) and terminal dexoynucleotidyl transferase-mediated deoxyurinetriphate (dUTP)-digoxigenin nick end labeling (TUNEL; Beyotime). The slides were imaged under a light microscope, and Cx43 levels in human biopsy specimens were analyzed using ImageProPlus software (Media Cybernetics, Silver Springs, MD, USA).
Cx43-modified BMSCs were cultured in a 6-well plate at 1 × 10⁵/well and fixed with 40 mg/L paraformaldehyde (Sigma) after cell adherence. Then, the cells were stained with anti-Cx43 (Santa Cruz) overnight at 4°C. After washing for three times, the cells were incubated with Anti-mouse Immunoglobin G (IgG; CST) for 30 min. Finally, the cells were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Boster) for 15 min, and the slides were sealed using 60% buffered glycerol (Sigma−Aldrich) and imaged under an immunofluorescence microscope (Olympus).

In immunohistochemical staining, 5‐µm‐sections were incubated for overnight in a 60℃ incubator to dissolve excess paraffin. Then, the tissues kept in xylene were passed through the decreasing alcohol series for rehydration and washed with distilled water and PBS, respectively. 3% hydrogen peroxide was added for the endogenous peroxide inactivation. Then, sections were incubated with 0.5% trypsin at 37℃ for 15 minutes at room temperature. Blocking solution was added onto the sections washed with PBS. Sections were incubated with primary antibodies anti‐NOS2 (sc‐7271, Santa Cruz), anti Cx43 (sc‐271837, Santa Cruz), and anti‐Caspase3 (sc‐56053, Santa Cruz) at +4℃ for overnight. The next morning, the tissues were washed with PBS, and biotinylated secondary antibody was added and waited for 10 minutes. The tissues were then washed three times with PBS again, and streptavidin peroxidase was added. After this step, the tissues were washed three times with PBS again and incubated with DAB to observe the immune reaction, and counterstaining was performed with Mayer's hematoxylin. Finally, the sections were washed with distilled water, passed through the increasing alcohol series, and covered with entellan. For all antibodies, tissues were evaluated considering the staining intensity in 10 ovary sections of each animal, in 30 areas in the X40 objectives.