Immunohistochemistry of TNFα, TNFAIP3, and IκBα

Formalin-fixed tissue was bundled using methods previously described [27 (link)]. Briefly, bundles were processed, embedded in paraffin wax in the transverse orientation, and 4 µm thick tissue sections were cut by microtomy and processed for immunohistochemistry (IHC). Tissue sections were de-paraffinised and rehydrated, then treated with 1% v/v hydrogen peroxide in methanol to block endogenous peroxidases, followed by heat-induced antigen retrieval in 0.01 M citrate acid buffer (pH 6.0). Sections were then blocked with 5% v/v serum supplied by respective ImmPRESS kits (Vector Laboratories, Heyford, UK), followed by primary antibodies against TNFα (rat monoclonal antibody MP6-XT22, Abcam; Cambridge, UK), TNFAIP3 (mouse monoclonal antibody 66695-1-Ig, Proteintech; Manchester, UK), and IκBα (rabbit polyclonal antibody 9242, Cell Signaling Technology; Leiden, The Netherlands). Secondary antibodies were from ImmPRESS polymer detector kits, and for TNFAIP3, the use of a mouse-on-mouse ImmPRESS kit. Slides were counterstained with haematoxylin (Vector Laboratories).