Dab substrate kit

H&E staining and IHC staining were performed as previously described^{65 (link)}. Briefly, after deparaffinization and rehydration, 4 μm paraffin-embedded tumor tissue sections were counterstained with H&E (ZSGB-BIO) and then dehydrated with graded alcohol and clearing in xylene. For IHC, after deparaffinization and rehydration, 4 μm paraffin-embedded tumor tissue sections were retrieved by citrate acid solution (pH 6.0) for 5 min at high pressure. The slides were subsequently incubated in 3% hydrogen peroxide and in goat serum. Primary antibodies against CD3ε (1:200, Cell Signaling Technology, Cat 99940), CD8α (1:400, Cell Signaling Technology, Cat 98941), Foxp3 (1:400, Cell Signaling Technology, Cat 12653), granzyme B (1:200, Cell Signaling Technology, Cat 44153), and HIF-1α (1:150, Novus, Cat NB100-105) were used. HRP-conjugated secondary antibodies (undiluted, anti-rabbit/anti-mouse, Cell Signaling Technology, Cat 8114/8125) were then used. The signals were detected by DAB substrate kit (Cell Signaling Technology). Slides were scanned using an Aperio ScanScope CS scanner (Leica). CaseViewer v. 2.4 (3DHISTECH) was used for creating slide presentations. Aperio ImageScope software v. 12.3.2 (Leica) was used for histoscore analysis of these detection indicators. The histoscore was normalized to 0–300.

Livers and other organs were dissected and embedded in OCT. Cryostat sections were cut at 10 μm. The sections were stored at −80 °C and subjected to hematoxylin/eosin and oil red O staining following standard protocol. The immunohistochemistry (IHC) staining of p62 was performed following the protocol of Cell Signaling Technology. The primary antibody was from Abcam, and the SignalStain® boost IHC detection reagent and DAB substrate kit were from Cell Signaling Technology. All the sections were imaged using a NanoZoomer 2.0-HT Digital slide scanner (Hamamatzu) and processed using NDP viewer software.

Formalin-fixed, paraffin-embedded lung sections (3 µm thick) from healthy donors and COPD patients were deparaffinized in xylene and rehydrated in a graded ethanol series. Heat-induced epitope retrieval was performed by boiling the slides in citrate buffer. After cooling at room temperature, the tissue sections were incubated with 3% hydrogen peroxide for 20 min, and then the nonspecific sites were blocked with 10% goat serum for 30 min. The slides were then incubated with antibodies for NOX1 (1:100, Novus Biologicals, Littleton, CO, USA), NOX2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), NOX4 (1:200, Novus Biologicals), or NOX5 (1:1000, Abcam, Cambridge, UK) in a humidified chamber at 4 °C overnight and washed three times with PBS. The slides were incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies, rinsed in PBS, and stained using the DAB Substrate Kit (Cell Signaling Technology, Danvers, MA, USA) with counterstaining of hematoxylin. The intensities of each NOX isoform in the lung sections were quantified using randomly selected images showing structures of alveolars, small airways, and pulmonary arteries in ten fields per subject with the Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA).