Streptavidin peroxidase conjugate

Paraffin sections of lungs were deparaffinized in xylene, and hydrated through graded alcohols. To stain for IL-33, heat-induced epitope retrieval was performed using 10 mM citrate buffer and, then, sections were placed in 0.05 M Tris-HCl-Tween buffer. We blocked endogenous peroxidase with 3% H₂O₂ in distilled water, and proteins with 1% BSA. Sections were then incubated with anti-IL-33 Ab (1∶50; monoclonal biotinylated Ab, Nessy-1, Enzo Life Sciences) in Tris+Tween+saponin buffer for 1 h. Finally, we used streptavidin/peroxidase conjugate (Dako) and prepared DAB chromogenic substrate. To stain for pro-SPC, endogenous peroxidase was blocked with 3% H₂O₂ in methanol, with HCl added last. Proteinase K (Dako) was used to unmask antigens/epitopes, and proteins were blocked with 5% normal goat serum. Sections were incubated with anti-pro-SPC Ab (1∶2000; polyclonal Ab, Millipore) in Ultra Clean Diluent (Fisher Scientific) for 1 h. Finally, we used EnVision detection system with DAB+ (Dako). Mayer’s hematoxylin was used to counterstain. Negative controls were generated by omitting the primary Ab. For enumeration of IL-33⁺ cells, counts were made from five fields of view per mouse (2 from the upper lobe, 2 from the middle and 1 from the lower respiratory tract) at 400× and normalized per unit area (mm²).

Immunohistochemistry with the tumour tissues collected from the xenograft study was performed as previously described (Lokman et al., 2013). Briefly, the formalin‐fixed tumour tissues were processed, embedded in paraffin and then sectioned (5 μm). The tissues then underwent antigen retrieval (5 min at 750 W, 15 min at 350 W in a microwave) in 10 mm citrate buffer (pH 6.5). The tissues were immunostained with Ki67 (rabbit monoclonal, 1/400; Epitomics, Burlingame, CA, USA) as described previously (Ricciardelli et al., 2011). Streptavidin‐peroxidase conjugate (1/500; Dako) and diaminobenzidine tetrahydrochloride (DAB; Sigma‐Aldrich) were used to visualise the staining. Images were recorded using a NanoZoomer Digital Pathology System (Hamamatsu Photonics, SZK, Japan).