Dewaxing, rehydration, and antigen retrieval was performed using a combined 3-in-1 Dako PT link module (Dako), and immunohistochemistry was performed manually by LH. Endogenous peroxidase activity was inhibited using EnVision FLEX Peroxidase-Blocking Reagent (Agilent Dako) for 60 minutes. Primary antibodies were incubated for one hour at room temperature: Ki67 at 1:400 (mouse monoclonal anti-Ki67, clone MIB-1, Cat no. M7240, Agilent Dako); γH2AX at 1:1000 (mouse monoclonal anti-gamma H2AX (phospho-S139) antibody, clone 9F3, Cat no. Ab26350, Abcam); and p21 at 1:100 (mouse monoclonal anti-p21, clone SX118, Cat no. M7202, Agilent Dako). For samples incubated with p21 antibody, an additional step involving 15 minutes incubation with EnVision FLEX mouse linker (Agilent Dako) was found to be optimal. An HRP-conjugated secondary antibody was applied for 30 minutes at room temperature (Dako EnVision FLEX/HRP, Agilent Dako), followed by incubation for five minutes with 3,3’-diaminobenzidine chromogen (Agilent Dako) and counterstain with Mayer’s haematoxylin for five minutes. Sections of canine skin were used as a positive control for each antibody (for example, as in (Kortum et al. 2018 (link))), and negative control slides were prepared using commercial species-matched immunoglobulins (Agilent Dako).
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