Sp 8400

Murine paraffin-embedded blocks were prepared from lungs fixed via the trachea with 10% buffered formalin at 20 cm H₂O. Serial sections were stained with anti-Ly6G clone 1A8 (ab25377, Abcam) and the following polyclonal antibodies: anti-SEMA3F (SAB2700501, Sigma-Aldrich), NRP1 (AF3870, R&D Systems), NRP2 (H300, Santa Cruz Biotechnology), or an isotype control following deparaffinization. Lung sections from patients with COPD undergoing resection for suspected lung tumor were stained with anti-SEMA3F, NRP2, or isotype control, and visualized with DAB or stained with anti-CD66b (555723, BD Biosciences), anti-NRP2 (HPA054974, Atlas Antibodies), and DAPI (422801, Sigma-Aldrich) with TSA plus system amplification (NEL744B001KT, Perkin Elmer) and autofluorescence quenching with TrueView (Vector, SP-8400).

The paraffin-embedded sections were deparaffinized and rehydrated as described in section 2.6. The sections were incubated with proteinase K working solution at 37℃ for 30 min and permeabilized with 0.1% Triton X-100 at room temperature for 20 min. Terminal deoxynucleotidyl transferase, dUTP, and buffer were mixed at a ratio of 1:5:50 and incubated with the sections at 37℃ for 2 h. The sections were then blocked with 3% BSA for 30 min and incubated with caspase-1 p10 (1:200) primary antibody overnight at 4℃. After washing, an Alexa Fluor594-conjugated secondary antibody (1:500) was added and incubated at room temperature for 2 h. An autofluorescence quenching kit (SP-8400, Vector Laboratories) was used to quench tissue autofluorescence according to the manufacturer’s instructions, and the nuclei were stained with DAPI. The sections were photographed under a fluorescence microscope.