Anti p histone h3 ser 10 antibody

Formalin-fixed tissue sections were embedded in paraffin, sectioned, deparaffinized, and blocked with 3% hydrogen peroxide in methanol. Antigen retrieval was performed using Diva Decloaking solution (Biocare Medical, Concord, CA) (120°C for 5 minutes followed by 100°C for 10 minutes). Slides were blocked with avidin-pink and biotin-blue (Biocare Medical), labeled with anti-p-Histone H3 (Ser 10) antibody (Cell Signaling Technology; Danvers, MA) in DaVinci Green (Biocare Medical), stored overnight at 4°C, and visualized with biotinylated goat anti-rat IgG (Accurate Chemical; Westbury, NY), followed by streptavidin-horseradish peroxidase (HRP; Invitrogen, Camarillo, CA) and diaminobenzidine (DAB; Sigma-Aldrich, St Louis, MO), and hematoxylin counterstaining. The number of positively-staining p-Histone-H3 (Ser 10) crypt enterocytes and the total number of cells per crypt were counted from at least 20 well-oriented crypts by blinded scoring. A proliferative index was calculated from the ratio of these measurements.

Paraffin-embedded sections were deparaffinized then blocked with 3% hydrogen peroxide in methanol. Diva Decloaking solution (Biocare Medical, Concord, CA) was used for antigen retrieval followed by blocking with avidin-pink and biotin-blue (Biocare Medical). Slides were then labeled with anti-p-Histone H3 (Ser 10) antibody (Cell Signaling Technology; Danvers, MA) or cleaved caspase 3 (CC3) (Cell Signaling Technology; Danvers, MA) in DaVinci Green (Biocare Medical) then incubated overnight at 4°C. Labeling was visualized with biotinylated goat anti-rat IgG (Accurate Chemical; Westbury, NY), then streptavidin-horseradish peroxidase (HRP; Invitrogen, Camarillo, CA) followed by diaminobenzidine (DAB; Sigma-Aldrich, St Louis, MO), and finally hematoxylin counterstaining. Positively staining p-Histone-H3 (Ser 10) or CC3 enterocytes were counted from at least 20 well-oriented crypts by blinded scoring. Apoptosis was scored on both CC3 and morphology using an H&E stain. For H&E morphology, apoptotic bodies were identified based on pyknosis, eosinophylic cytoplasm, and perinuclear halo in twenty well-formed crypts per animal.