Mouse anti cd68 clone kp1

Human paraffin-embedded gingival tissue samples were collected from PD patients. Five-micrometre sections were prepared on glass slides. Before staining, endogenous peroxidase activity was inhibited by incubating 0.3% H 2 O 2 in methanol and followed by blocking off non-specific antibody binding with 1% BSA and 1% normal goat serum in PBS. Subsequently, tissue samples were stained with rabbit anticitrullinated histone H3 (ab5103; Abcam, Cambridge, UK) 1:250 in PBS + 1% BSA, rabbit anti-PAD4 (ab3877; Abcam) 1:200 in PBS + 1% BSA or mouse anti-CD68 (clone KP1; Dako, Glostrup, Denmark) 1:100 in PBS + 1% BSA. Next, sections were incubated with goat anti-rabbit IgG-HRP (P0448; Dako) in PBS + 1% BSA or rabbit anti-mouse IgG-HRP (P0260; Dako), followed by using a DAB kit (K3467; Dako). Tissues were additionally stained with a rabbit IgG control (Southern Biotech, Birmingham, AL, USA) to control for non-specific binding, as previously described (Nesse et al., 2012) . Sections were counterstained with haematoxylin and mounted with glycerin. In each periodontal tissue sample, positive cells were counted in 10 adjacent fields with a 40× objective (magnification 400×). Mean cell numbers per tissue sample were calculated for each staining. Immunohistochemical scoring was performed by one trained person.