Ab4044

Cells were lysed with RIPA lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics) and fractionated using the NE-PER Nuclear and Cytoplasmic extraction reagents (Thermo Scientific). Supernatants were filtered and concentrated by trichloroacetic acid precipitation, followed by aceton desiccation. Proteins were run on SDS-PAGE (4–12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes (GE Healthcare) blocked with 5% bovine serum albumin (BSA) or non-fat dry milk (NFDM), incubated with primary antibody: rabbit anti-IL-1 beta (1:2,500 in 5% NFDM, ab9722, Abcam), rabbit anti-ASC (1:200 in 5% BSA, sc-22514-R, Santa Cruz), mouse anti-NLRP3/NALP3 (1:1,000 in 5% milk, Cryo-2, Adipogen) or rabbit anti-MMP7 (1:200 in 5% BSA, ab4044, Abcam), washed with PBS tween 0.1% and incubated with secondary antibodies in 5% NFDM (goat anti rabbit-HRP or goat anti-mouse-HRP, Cell Signaling). Bands were imaged using ECL Plus detection reagent (GE Health Care) and quantified using ImageJ. GAPDH (1:1,000, sc-25778, Santa Cruz) was used as loading control.

Tissue arrays were each made from 25 to 30 collections of paraffin-embedded tissue cores (1 mm diameter) and sections (5 µM) cut for antibody labelling. A BondmaX™ automated system (Leica BioSystems, Seoul, Korea) was an automated staining platform [18 (link),19 (link)], and antibodies were optimised for concentration, pH-dependence and antigen retrieval. Those used, as per manufacturer’s instructions, were: β-catenin (ab22656, Abcam, Cambridge, UK), c-Myc (Novacastra/Leica Biosystems), cyclin-D1 (sc 718, Santa Cruz, Santa Cruz, CA, USA) and MMP-7 (ab4044, Abcam). The researcher was blinded to the tissue array arrangement. Fluorescent labels were used: FITC (488/517 nm, green, β-catenin), Cy3.5 (561/617 nm, red, c-Myc), Cy5 (633/671 nm, purple, cyclin-D1) and Cy3 (514/565 nm, yellow, MMP-7). For expression analyses, each section was imaged with a TCS SP8 confocal system (Leica) at 63x magnification. Then, from five regions under a further 6x times digital magnification, the number of pixels at each fluorophore channel was measured using a macro compiled within Huygens Professional software (SVI, Hilversum, The Netherlands) to measure individual channel intensities [18 (link)].

Tissues were embedded in O.C.T. compound (VWR) and 5-μm-thick fresh cryosections on positively charged microscope slides (Superfrost/Plus; Thermo Scientific) were fixed with 4% paraformaldehyde or acetone-methanol (1:1 v/v). For H&E or immunohistochemistry, sections were blocked and permeabilized (0.2% Triton X-100, 5% goat normal serum (DAKO) or 1% BSA (Sigma), stained (anti-neutrophil antibody [NIMP-R14] (ab2557, Abcam), polyclonal E. coli antibody (1:100, NB200-579, Novus Biologicals), anti-IL-1 beta (1:50, ab9722, Abcam) or anti-MMP-7 (1:100, ab4044, Abcam), all rabbit antibodies). Alexa 488 anti-rat IgG or anti-rabbit IgG and Alexa 568 anti-rabbit IgG (A-21210, A-11001 and A-11011, Life Technologies) were secondary antibodies and nuclei were counterstained with DAPI (0.05 mM, Sigma-Aldrich). Imaging was by fluorescence microscopy (AX60, Olympus Optical). Richard-Allan Scientific Signature Series Hematoxylin 7211 and Eosin-Y 7111 (Thermo Scientific) were used to counterstain the tissue sections.
Histology was scored using H&E stained bladder sections. The score was based on neutrophil infiltration, tissue architecture and epithelial thickness on a scale of 0–10, where 0 is unchanged compared to uninfected controls and 10 the highest neutrophil infiltration, most destroyed tissue architecture and maximum epithelial thickness.