Immunohistochemistry for the paraffin sections was carried out using rabbit anti-caspase 3 (dilution 1:500; Cat. No. NB100–56113, Novus Biologicals, Littleton, CO, USA) as a marker for apoptosis [64 (link)], rabbit anti-TNF-α (dilution 1:200; Cat. No. NB600–587, Novus Biologicals, Littleton, CO, USA) and mouse anti-interleukin-6 (IL-6) (dilution 1:400; Cat. No. ab9324, Abcam, Cambridge, UK) as markers of inflammation [47 (link)]. Antigen retrieval was performed by heating in citrate buffer then quenching of endogenous peroxidase was processed using a peroxidase blocker. The sections were incubated with the primary antibodies (Ab) in a humidity chamber overnight followed by incubation with the secondary Ab for 45 min. Visualization of the reaction was performed using a 3,3′-Diaminobenzidine (DAB) detection kit with counterstain by Meyer hematoxylin. The area % of the (+ve) immunostaining for caspase3, TNF-α and IL-6 was measured using Leica Qwin 500 C image analyzer. The measures were obtained from 10 non-overlapping low power fields/section in each group.
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