Normal goat serum (ngs)

TSCC and paired adjacent tissue specimens from enrolled patients were acquired from the Pathological Department of the aforementioned hospital; the samples were paraffin-embedded. Then, immunohistochemistry was performed to assess ITGA7 and CD133 expression in tumor and paired adjacent normal tissues. Briefly, paraffin-embedded tissue sections (4-μm thick) were deparaffinized with xylene and rehydrated with ethanol. Heat-induced antigen retrieval was subsequently conducted in 0.01 mol/l sodium citrate buffer (pH 6.0) using a microwave, and endogenous peroxidase activity was inhibited with freshly prepared 3% H₂O₂. Following blocking using 1.5% normal goat serum (Shanghai Yeasen Biotechnology Co., Ltd.) at 37°C for 20 min, the sections were incubated at 4°C overnight with rabbit polyclonal ITGA7 antibody (1:200; cat. no. ab203254; Abcam) and rabbit polyclonal CD133 antibody (1:100; cat. no. ab19898; Abcam). The next day, tissue sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (diluted 1:1,000 in 3% bovine serum albumin; cat. no. ab6721; Abcam) at 37°C for 90 min. Subsequently, 3,3′-diaminobenzidine and hematoxylin were applied for staining of the sections, which were then sealed with neutral tree gum. ITGA7 and CD133 expression was evaluated under a light microscope.

Mice lung samples were freshly isolated and fixed in 10% neutral buffered formalin and then embedded in paraffin wax. Lung sections with a thickness of 4 μm were mounted onto slides. Slides were deparaffinized with xylene, rehydrated with ethanol, and incubated with H₂O₂ at 37°C for 10 minutes. Following blocking using 1.5% normal goat serum (Shanghai Yeasen Biotechnology Co., Ltd.) at 37°C for 20 minutes, sections were incubated overnight with Ki-67 or NF-κB p65 monoclonal antibody (1:1000 dilutions). The sections were incubated with biotin-conjugated goat anti-rabbit immunoglobulin G secondary antibody (diluted with 3% bovine serum albumin/PBS) at 37°C for 30 minutes and then incubated with horseradish peroxidase–conjugated streptavidin at 37°C for 30 minutes. DAB was used as chromogenic agent. Images were obtained using a fluorescence microscope (FSX100; Olympus, Southend-on-Sea, UK).

The tumor tissues and adjacent noncancerous pulmonary tissues were freshly isolated from surgery, then fixed in 10% neutral‐buffered formalin, and then embedded in paraffin wax. The level of AKIP1 in the paraffin‐embedded specimens was assessed by IHC assay as follows: The specimens were cut into 4‐µm sections, deparaffinized with xylene, rehydrated with ethanol followed by antigen retrieval, and incubated with H₂O₂ for endogenous peroxidase blocking, then were blocked using 1.5% normal goat serum (Yeasen Biotechnology Co., Ltd). Subsequently, the tissue sections were incubated at 4℃ with rabbit‐anti‐C11orf17/AKIP1 antibody overnight (1:100 dilution) (Abcam). Next day, the tissue sections were incubated with horseradish peroxidase‐conjugated goat‐anti‐rabbit immunoglobulin G antibody (1:1000 dilution) (Abcam) for 30 minutes. Afterward, tissue sections were stained with diaminobenzidine (DAB) and hematoxylin and were sealed with neutral tree gum. Finally, the positive cells were observed and counted using a fluorescence microscope (Olympu0073).

Lung samples were freshly isolated and fixed in 10% neutral buffered formalin and then embedded in paraffin wax. Lung sections with a thickness of 4 μm were mounted onto slides. Slides were deparaffinized with xylene, rehydrated with ethanol, and incubated with H₂O₂ at 37°C for 10 minutes. Following blocking using 1.5% normal goat serum (Shanghai Yeasen Biotechnology Co., Ltd.) at 37°C for 20 minutes, sections were incubated overnight with αvβ6 or IL-8 monoclonal antibody (1:1000 dilutions). The sections were incubated with biotin-conjugated goat-anti-rabbit immunoglobulin G secondary antibody (diluted with 3% bovine serum albumin/PBS) at 37°C for 30 minutes and then incubated with horseradish peroxidase–conjugated streptavidin at 37°C for 30 minutes. 3,3’-Diaminobenzidine (DAB) was used as chromogenic agent. Images were obtained using a fluorescence microscope (FSX100; Olympus, Southend-on-Sea, UK).

Sections with a thickness of 4 µm were mounted onto slides that were coated with adhesive. Slides were deparaffinized with xylene, rehydrated with graded concentrations of ethanol, and incubated with H₂O₂ at 37°C for 10 min. Then, the sections were washed with phosphate-buffered saline (PBS) and heat-mediated antigen retrieval was performed using a microwave. Following blocking using 5% (v/v) normal goat serum (Shanghai Yeasen Biotechnology Co., Ltd.) at 37°C for 10 min, sections were incubated overnight with NF-κB p105/p50 monoclonal antibody (ab32360; Abcam) at a dilution of 1:1,000. Washing with PBS was completed three times and the sections were incubated with biotin-conjugated goat-anti-rabbit immunoglobulin G secondary antibody (diluted with 3% bovine serum albumin/PBS; ab64257; Abcam) at 37°C for 30 min. Further washing with PBS of the sections was completed three times, prior to incubation with horseradish peroxidase-conjugated streptavidin at 37°C for 30 min. The sections were then washed again using PBS three times and 3,3′-diaminobenzidine (DAB) was used as chromogenic agent. The streptavidin-peroxidase IHC kit was purchased from Maxim Biotech, Inc. (Rockville, MD, USA).

For immunohistochemical procedures, spleen sections (n = 3 per group) were air-dried, fixed, heated with citrate buffer (10 mM, pH 6.0) for antigen retrieval, and blocked with 5% normal goat serum (Yeasen, Shanghai, China). Sections were then incubated with anti-p-NFκB (AF2006; 1:100; Affinity, Changzhou, China), anti-iNOS (AF0199; 1:100; Affinity, Changzhou, China), or anti-Cox-2 (AF7003; 1:100; Affinity, Changzhou, China) overnight at 4 °C followed by incubation with HRP conjugated secondary antibody (S0001; 1:200; Affinity, Changzhou, China) for 1 h at room temperature. Then, the target proteins in spleen sections were visualized using a DAB kit (ZSGB-Bio, Beijing, China) and the nuclei were counterstained with hematoxylin (Leagene, Beijing, China). After sealing, the slides were scanned by using a EasyScan Pro 6 (Motic, Xiamen, China).