Bond max 2

Tissue samples were fixed in 10% formalin, paraffin-embedded and cut in 3-μm sections, which were mounted in superfrostplus microscope slides (Thermo Scientific, Cat. 165061) and dried. The immunohistochemistry was performed using an automated immunostaining platform (Ventana discovery XT, Bond Max II, Leica). Antigen retrieval was performed with low pH buffer (CC1m) for p-H2AX and high pH buffer (ER2) for BrdU. Endogenous peroxidase was blocked (peroxide hydrogen at 3%), and slides were then incubated with anti-BrdU (BU-1, GE Healthcare, RPN202, Lot. 341585, 1:100) and phospho-histone H2AX (Ser139) (γH2AX, JBW301, Millipore, 05-636, Lot. DAM1493341, 1:4000). After the primary antibody, slides were incubated with the corresponding secondary antibodies when needed (rabbit anti-mouse Abcam) and visualization systems (Omni Map anti-Rabbit, Ventana, Roche; Bond Polymer Refine Detection, Bond, Leica) conjugated with horseradish peroxidase. Immunohistochemical reaction was developed using 3,30- diaminobenzidine tetrahydrochloride (DAB) (ChromoMap DAB, Ventana, Roche; Bond Polymer Refine Detection, Bond, Leica), and nuclei were counterstained with Carazzi’s hematoxylin. Finally, the slides were dehydrated, cleared, and mounted with a permanent mounting medium for microscopic evaluation.