N histofine kit

Sections were incubated with anti-ATM (Invitrogen, Rockford, IL, USA), anti-αSMA (DAKO A/S, Glostrup, Denmark) or anti–surfactant-B Ab (Merck, Darmstadt, Germany), reincubated with biotinylated anti-mouse, or anti-rabbit Ab, streptavidin–biotin complex, peroxidase substrate (N-Histofine kit, Nichirei Biosciences, Tokyo, Japan) and counterstained with hematoxylin [19 (link)].

Double perforin/CD56 and perforin/CD3 immunostainings were performed on consecutive 4 µm thick liver sections (n = 4) from formalin-fixed, paraffin-embedded tissue, as described in [33] (link). Slides were first immunostained with the anti-perforin Ab2 antibody (Clone 5B10, Neomarkers) and revealed with the N-Histofine kit (Nichirei Biosciences Inc). Fast red was used as the chromogen for perforin immunodetection. Then, antigen retrieval was performed in Tris-citrate buffer for CD56 or citrate buffer for CD3. Slides were incubated with the primary anti-CD56 (clone 1B6, Novocastra) or anti-CD3 (polyclonal rabbit CD3, Dako) antibodies and then incubated with the secondary antibody using the Envision kit. DAB was used as the chromogen for CD56 or CD3 immunodetection. All sections were counterstained with Harris hematoxylin, washed in tap water and mounted in aquamount (Merck). Sur-fixation in glutaraldehyde (0.05% in PBS pH 8.6) followed each incubation with the primary or secondary antibody.