Avidin blocking solution

Tissue sections were deparaffinized and rehydrated, endogenous peroxidase activity neutralized and antigens retrieved by boiling in sodium citrate solution (Vector Labs H-3300). Sections were blocked in 2% bovine serum albumin (BSA)/10% normal serum (same species of secondary antibody) and avidin-blocking solution (Vector) in phosphate-buffered saline (PBS), incubated with primary antibody in 2% BSA/10% normal serum and biotin-blocking solution (Vector) in PBS, incubated with biotinylated secondary antibody 1:500 (Vector), incubated with ABC (Vector PK-6100) or ABC-AP reagent (Vector AK-5000), incubated with DAB/substrate/chromogen system (Dako K3467; brown) or AP substrate kit I (Vector SK-5100 reagent, red) and counterstained with hematoxylin (Vector H-3404). Specificity of staining was confirmed by omission of the primary antibody. Images were obtained using a digital microscope (COOLSCOPE, Nikon). Primary antibodies used included anti-β-catenin (Santa Cruz sc-7963, 1:250 dilution), anti-α-SMA (Sigma A2547, 1:200 dilution), anti-Sm22α (Abcam 14106, 1:500 dilution), anti-CD31 (Abcam 28364, 1:50 dilution) and anti-pHH3 (Cell Signaling 9701, 1:50 dilution).

Thawed sections were fixed with paraformaldehyde (4%, 20 min), washed in PBS (2 × 5 min), incubated in pre‐chilled 3% H₂O₂ in methanol (20 min), and then washed in PBS (2 × 5 min). Each section was blocked with normal goat serum (NGS; 10%; 30 min; #S‐1000; Vector Laboratories, CA, USA), incubated with AVIDIN blocking solution (15 min; #SP‐2001; Vector Laboratories), rinsed in PBS, and then incubated with rat anti‐mouse CD68 primary antibody (1:200; 5% NGS; 4°C; #mca1957ga; Bio‐Rad, NSW, Australia) overnight. The slides were then washed in PBS (2 × 5 min) before being incubated with the secondary antibody (1:100; 5% NGS; 30 min; #BA‐4000; Vector Laboratories). Next, the sections were washed in PBS (2 x 5 min) and incubated with ABC avidin/biotin complex (30 min; PK‐6100; Vector Laboratories) and DAB solution (#SK‐4100; Vector Laboratories). Staining was terminated with distilled water. The sections were counterstained with Mayer Haematoxylin for 15 s and rinsed in tap water before blueing in Scotts tap water and washing in tap water. Finally, slides were dehydrated in ethanol (95% 3 min, 100% 3 × 3 min), cleared in xylene (2 × 5 min) and mounted with depex (#13515; VWR International, USA). Sections were imaged on the Olympus FSX100 microscope 4.2 × magnification, and images were analysed using Adobe Photoshop CC.

Immunohistochemistry of neutrophils in lung sections was performed with the Vectastain ABC Kit (Linaris, Mannheim, Germany). After blocking with avidin blocking solution (Vector Labs, Burlingame, USA) for 1 h at room temperature, sections were incubated with a monoclonal rat Ly-6B.2 Alloantigen Antibody 7/4 (AbD Serotec, Cat. No. MCA771GA) or rat IgG control (Santa Cruz, Cat. No. sc-2026) at a dilution of 1:1000 overnight at 4°C. Tissue sections were then incubated with biotinylated rabbit anti-rat IgG (Vector Labs, Cat. No. BA-4000) for 30 min followed by Vectastain ABC reagent (Vector Labs, Cat. No. PK-4000) for 30 min and then developed using Histogreen (Liniaris, Cat. No. E109) as substrate. Counterstaining was performed using nuclear fast red (Linaris, Cat. No. H-3403). Images were then processed with a Leitz DM IRB microscope (Leica). All images were analyzed with the software AxioVision v4.8.2