Dapi g1012

Immunofluorescence staining for expression of γ‐H2AX was conducted as described in a previous study (Xu, Xu, et al., 2019 (link)). Briefly, sections cut from the testes in different groups were incubated with 5% fetal bovine serum for 30 min at room temperature and then incubated overnight at 4°C with primary antibodies against γ‐H2AX (ab81299, Abcam, 1:200). Goat anti‐mouse Alexa Fluor 488 (A0428, Beyotime, CHN) was used for secondary staining. Nuclei were counterstained with DAPI (G1012, Servicebio, CHN). Images of the stained samples were captured using a fluorescence microscope (Olympus, Tokyo, Japan). The γH2AX expression was analyzed from the images of IF staining by using ImageJ software 1.8.0 (National Institutes of Health, United States). The data were expressed as a percentage of the fluorescence of the control group.

The lectin‐histochemical study was performed on pig uterine cross sections. Slides were incubated with either fluorescein isothiocyanate (FITC) labelled lectin Sambucus nigra (SNA, 1:150, FL‐1391; Vector Labs) or biotin‐conjugated lectin Maackia amurensis (MAL‐II, 1:150, B‐1265; Vector Labs) at 4°C overnight. The biotin‐conjugated lectin was subsequently detected with streptavidin‐FITC conjugate (1:100, SA‐5001; Vector Labs). Nuclei were stained with DAPI (G1012; Servicebio) and sealed with anti‐fluorescence quenching tablets. All slides were scanned by a Pannoramic Midi slide scanner (3D HISTECH), and the image analysis was carried out by using CaseViewer 2.0 software (3D HISTECH).

NCI-H460 cells were seeded in 24-well plate (with coverslips plated) at the density of 5 × 10⁴ cells. After overnight adherence, the cells were exposed to compounds at 5 nM or 10 nM for 24 h, respectively. Then, cells were fixed with 4% cold paraformaldehyde at 4 °C for 15 min and then permeabilized in 0.05% Triton X-100 for 10 min. The cells were blocked in 1% BSA for 30 min. Microtubules were detected by incubation with a monoclonal anti-β-tubulin (β-tubulin antibody was obtained from Servicebio, Wuhan, China.) at 4 °C for 12 h. Subsequently, the cells were washed with PBS and incubated with a FITC-conjugated anti-mouse IgG antibody. Nuclei were stained with DAPI (G1012, Servicebio, Wuhan, China). The coverslips were visualized under fluorescence microscope (Nikon Eclipse C1, Nikon, Japan) and an image-forming system (Nikon DS-U3, Nikon, Tokyo, Japan) [17 (link),21 (link)].

After obtaining the patient’s consent and approval from the Ethics Committee of Qingdao Central Hospital (KY202318401), we obtained pathological sections from KIRC patients. Anti-LAG3 antibody (ab209236, 1:100 dilution), anti-CD45 antibody (ab40763, 1:100 dilution), and anti-PAX8 antibody (ab191870, 1:1000 dilution) were purchased from Abcam (Cambridge, UK). BSA (G5001), PBS (G0002), EDTA (G1203), and DAPI (G1012) were purchased from Servicebio (Wuhan, China). Paraffin-embedded tissue sections underwent xylene deparaffinization, followed by ethanol dehydration. Microwave antigen retrieval (EDTA buffer, pH 9.0, 10 minutes) was performed, and non-specific binding was blocked with BSA for 30 minutes. Anti-CD45, anti-PAX8, and anti-LAG3 antibodies were applied and incubated overnight at 4°C. After three PBS washes, sections were exposed to secondary antibodies for 50 minutes at room temperature under light protection. DAPI staining (10 minutes) for cell nuclei and a 5-minute application of autofluorescence quenching reagent were followed. Sections were then mounted on slides for microscopy, and images were captured using a fluorescence microscope.

The mice PMVECs were isolated by magnetic-activated cell sorting. Briefly, the mice lung tissue was isolated under a sterile condition and marginal lung tissue was cut within 3 mm into small pieces. Then, the lung tissue was digested with type I collagenase solution (American Sigma) at 37°C for 45 min, before being centrifuged and filtered using 100 µm and 40 µm cell mesh. Next, CD31+ magnetic beads (Mildteny, Germany) were used and solutions were incubated at 4°C for 15 min. Finally, the cells were passed through an LS sorting column (Mildtenny, Germany) and centrifuged to obtain PMVECs. PMVECs are grown in 5% CO2 in endothelial cell culture medium at 37°C (Sciencell, USA). Higher purity endothelial cells were obtained after seven days.
After that, the PMVECs were identified by immunofluorescence staining with von Willebrand factor (vWF). Briefly, PMVECs were seeded in 24-well plates and blocked with 10% bovine serum albumin in 0.5% TritonX-100/PBS, before being incubated with the primary antibody of vWF (SA00006-4, Proteintech, USA) at 4°C for 12 h. Then, the cells were incubated with second antibody at room temperature for 2 h and stained with DAPI (G1012, Servicebio, China). Finally, fluoromount-G (0100-01, SourthernBiotech, USA) was added to the glass and a fluorescence microscope was used for analysis (OLYMPUS, Japan).

The EUB338 16S rRNA probe labeled with the fluorophore Cy3 (extinction wavelength, 555 nm; emission wavelength, 570 nm; Servicebio, China) was used to detect the bacterial colonization within mouse pancreatic tissues by FISH. Nuclei were stained with DAPI (G1012, Servicebio). Fluorescence microscopic analysis was conducted with Nikon Eclipse 90i confocal microscope (Nikon, Melville, NY) using a Cy3 labeled-probe at 1μM as described [51 (link)–53 ].