Ab203563

Lungs were collected and fixed in 4% buffered formaldehyde in PBS pH 7.2–7.4 (Bio Lab, Jerusalem, Israel) for 2 weeks. Sections of 5 µm were prepared after paraffin embedding using an RM 2255 microtome (Leica, Nussloch, Germany). Antigen retrieval was performed by incubation in Target Retrieval Solution (S1700, DAKO, Carpinteria, CA, USA, 30 min, 95 °C). After blocking in 5% BSA in PBS, slides were incubated (overnight, 4 °C) with purified anti-CD31 (390, Biolegend, San Diego, CA, USA), proSPC (Millipore, Temecula, CA, USA), podoplanin (T1α, 8.1.1, Biolegend), VE-cadherin (ab33168), claudin 5 (ab15106), connexin 43 (ab117843), occludin (ab31721) or claudin 18 (ab203563) (Abcam, Cambridge, MA, USA). Alexa Fluor 594- or 488-coupled donkey anti-rabbit or Alexa Fluor 594-coupled goat anti-Armenian hamster antibodies were used for detection (Molecular probes®, Thermo Fisher Scientific, Carlsbad, CA, USA). For nuclear staining, slides were mounted with Prolong® Gold antifade reagent containing DAPI (Molecular probes®, Thermo Fisher Scientific, Carlsbad, CA, USA). Analysis was performed using an LSM 710 confocal scanning microscope (Zeiss, Jena, Germany) equipped with the following lasers: argon multiline (458/488/514 nm), diode 405 nm, DPSS 561 nm and helium-neon 633 nm.

Serial sections of 4 mm thickness were taken from representative FFPE tissue blocks, affixed to 3‐aminopropyl triethoxysilane‐coated slides, and air‐dried overnight at 37°C. The sections were then subjected to HE and immunohistochemical staining. Immunohistochemical staining was performed using the following immunoperoxidase polymer methods. After dewaxing and antigen retrieval, endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 10 min. After blocking with goat serum, the sections were incubated for 30 min at 37°C with rabbit anti‐CLDN18 (1:500 dilution, clone ab203563; Abcam), rabbit anti‐CDX2 (1:50 dilution, clone A20222; ABclonal Technology Co), rabbit anti‐PAX8 (1:100 dilution, clone bs‐1201R; BIOSS ANTIBODIES), mouse anti‐CEA (1:300 dilution, clone 12‐140‐10; ZSGB‐BIO), mouse anti‐p53 (1:300 dilution, clone DO‐7; ZSGB‐BIO), and mouse anti‐P16 (1:100 dilution; clone MX007, MXB). The slides were visualized with diaminobenzidine (DAB) and counterstained with hematoxylin. Both membranous and nuclear CLDN18 levels were evaluated. The stained slides were scanned with a Pannoramic SCAN slide scanner (3DHISTECH Ltd.) to obtain a whole slide image.

The microporous membrane covered with HSAECs was cut from the upper chamber and placed into a 24-well plate. After 3 washes with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and washed again with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then washed 3 times with PBS again. The cells were then blocked in 5% goat serum for 60 min and incubated with rabbit primary antibodies against ZO-1 (Abcam, ab96587) at a 1:200 dilution, claudin-18 (Abcam, ab203563) at a 1:200 dilution, and occludin (Abcam, ab235986) at a 1:100 dilution at 4°C overnight. After washing with PBS, the cells were incubated for 60 min with Alexa Fluor^® 594-conjugated goat anti-rabbit IgG (ZF-0516, ZSGB-BIO, Beijing China) at a 1:200 dilution in the dark. After washing with PBS, membranes were embedded in 50% glycerol. Cells were visualized using a confocal microscope (TCS SP2, Leica, Germany). Representative images were obtained with a digital camera and then processed with Adobe Photoshop CS2.