Immunohistochemical analysis was performed as previously described at 4 days after DNBS administration [31 (link)]. The sections were incubated overnight with primary antibodies: anti-ICAM-1 mouse polyclonal antibody (1:100 in PBS, v/v, Santa Cruz Biotechnology SCB, D.B.A, Milan, Italy), anti-P-selectin mouse polyclonal antibody (1:100 in PBS, v/v, SCB, D.B.A, Milan, Italy), anti-PARP mouse polyclonal antibody (1:100 in PBS, v/v, SCB, D.B.A, Milan, Italy), anti-nitrotyrosine rabbit polyclonal antibody (1:200 in PBS, v/v, Millipore, D.B.A, Milan, Italy), anti-MPO (Neomarkers, 1:200 D.B.A, Milan, Italy). All sections were washed with PBS and then treated as previously reported [31 (link)].
Five stained sections from each mouse were scored in a blinded fashion and observed using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) following a typical procedure [36 (link)]. The histogram profile is related to the positive pixel intensity value obtained [37 (link)].
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