Anti podoplanin

Total protein was extracted from the lung tissue, and its concentration was determined using an enhanced BCA kit (Beyotime Biotechnology). Thereafter, 30 µg of the protein sample was subjected to 10% SDS-polyacrylamide gel electrophoresis, followed by transfer of the separated proteins to a polyvinylidene fluoride membrane (ISEQ00010, Millipore, USA) (26 (link)). After blocking with 5% skim milk at 37 °C for 2 h, the membrane was incubated with anti-claudin-18 (1:1,000, Abcam, USA), anti-claudin-4 (1:1,000, Invitrogen, USA), anti-SFTPC (1:1,000, Proteintech, China), anti-podoplanin (1:1,000, Santa Cruz, USA), anti-GSK-3β (1:1,000, CST,USA), anti-p-GSK-3β (1:1,000, CST, USA), and anti-β-catenin (1: 1,000, CST, USA) antibodies overnight at 4 °C. The next day, membranes were incubated with the corresponding horseradish peroxide-conjugated secondary antibody (S0001, S0002, Affinity) at 37 °C for 2 h. Amersham Imager 600 (GE Healthcare Life Sciences) was used to detect chemiluminescence signals, and the Image J 1.8.0 was used to calculate the gray value. The experiment was repeated at least thrice.

Immunohistochemistry of formalin-fixed, paraffin-embedded tissues from human explants was performed with the Histostain-Plus detection System according to the manufacturer's protocol (Life Technologies). Sections were dewaxed and rehydrated, and antigen retrieval was performed by heating the slides in 10 mM citric acid buffer (pH 6.0) for 15 min. Sections were incubated overnight at 4 °C in a humidified chamber with a 1 : 10 dilution of antibody to p16 (BD) and 1 : 50 NF2 (Santa Cruz). Control IHC experiments (data not shown) were performed without primary antibody. All sections were counterstained with Gill's haematoxylin and mounted for digital slide scanning.
For immunofluorescence, isolated cells were first fixed in 4% paraformaldehyde and where indicated permeabilized with 0.1% Triton in PBS-Tween (0.1% vol/vol), prior to blocking in 5% goat serum in PBS-Tween. Cells were then incubated with primary antibody 1 : 50 anti-Podoplanin (Santa Cruz) without prior permeabilization; 1 : 50 anti-Calretinin (Cell Signaling) overnight at 4 °C. Cells were washed three times for 10 min each with 1 × PBS, incubated for 1 h with secondary antibody (in blocking solution), washed three times with 1 × PBS, and counterstained with DAPI and mounted for confocal microscopy.