Gamma h2ax

The immunohistochemical detection of 8-Oxoguanine (oxidative DNA damage marker), gamma H2AX (DNA-double strand breaks), APE-1 and OGG1 (DNA repair enzymes), and cleaved caspase-3 (apoptosis) in placental sections from GDM and AnxA1^−/− mice was performed. Endogenous peroxide activity was blocked using 3% hydrogen peroxide for 30 min. The tissue sections were then incubated with the following primary antibodies overnight at 4 °C: 8-Oxoguanine (Abcam, Cambridge, UK), gamma H2AX (Novus Biologicals, Littleton, CO, USA), OGG1 (Novus Biologicals), APE-1 (Novus Biologicals), polyclonal rabbit anti-caspase-3 (Abcam). For negative control, sections were incubated with 10% TBS-BSA (Sigma–Aldrich, St. Louis, MO, USA) instead of the primary antibody. After washing with TBS, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam). Staining was visualized using a 3,3′-diaminobenzidine substrate (Invitrogen, Waltham, MA, USA). Finally, the sections were counterstained with hematoxylin (Inlab Confiança, São Paulo, Brazil) and mounted using Entellan (Merck, Germany).

Antibodies to KLF2 (Cat # ab203591), KLF4 (Cat # ab106629), and MEF2 (Cat # ab64644) were purchased from Abcam (Burlingame, CA, USA). Antibodies to TM (Cat # NBP1-95319), ICAM-1 (Cat # NBP2-22541), eNOS (Cat # NB300–500), E-selectin (Cat #NBP1-45545), and gamma H2AX (Cat # NB100-384) were procured from Novus Biologicals. Antibodies to ERK5 (Cat #3372), phosphoERK5 (Cat #3371), and β-actin (Cat #4970) were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA).
Anti-rabbit IgG-HRP (Cat #7074) and anti-mouse IgG-HRP (Cat #7076) secondary antibodies were purchased from Cell Signaling Technology Inc. Fluorochrome-conjugated goat anti-rabbit IgG (H + L)-Alexa Fluor 488 (Cat # A11034), donkey anti-goat IgG (H + L)-Alexa Fluor 568 (Cat # A11057), and goat anti-mouse IgG (H + L)-Alexa Fluor 488 (Cat # A11029) were procured from Invitrogen (ThermoFisher Scientific).