Ab135506

After the extraction of total protein with lysis buffer, the protein concentration was measured by Bicinchoninic acid (BCA) assay. Later, the extracted protein (50 μg) got electrophoresis under constant voltage in 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAG), followed by wet-transfer into a polyvinylidene fluoride (PVDF) membrane. The transferred membrane was blocked with 5% skim milk powder at room temperature, followed by the addition of primary antibodies (VEGF-C: ab135506, 1:500, Abcam, Cambridge, UK; VEGFR-3: ab51874, 1:800, Abcam, Cambridge, UK; LYVE-1: ab33682, Abcam, 1:100, Cambridge, UK). Then this sample was incubated overnight at 4°C on a shaking table and washed for 3 times, after which goat-anti-rabbit horseradish peroxidase conjugated antibodies were added (ab6721, Abcam, 1:2000, Cambridge, UK) as secondary antibodies. Subsequently, the sample was incubated at room temperature for 1 hour, followed by coloration with enhanced chemiluminescence (ECL) plus reagent and imaging. The calculation of the grey level of target bands was conducted with Synegene gel imaging system and GeneTools was employed for analyses on protein bands. β-actin was used as internal reference (ab194952, 1:1000, Abcam, Cambridge, UK) for adjustment.

The 4 μm thick, formalin‐fixed, paraffin‐embedded tumors of clinical specimens of lung adenocarcinoma were deparaffinized in xylene and rehydrated in a graded series of alcohols, then rinsed three times with PBS. Antigen retrieval was performed in the pressure cooker at 130°C for three minutes, citrate buffer (PH 6.0) was used for VEGFC staining, and EDTA solution (PH 11.0) was used for PD‐L1 staining. The slides were then incubated in 3% H₂O₂ for 15 minutes. For immunohistochemical staining the slides were incubated with primary antibodies against VEGFC (ab135506, Abcam, USA), 1:100, or against PD‐L1/CD274 (66 248, Proteintech, USA), 1:1200, at 4°C, overnight. Incubation of secondary antibody and coloration were then carried out by EIVISON plus (kit‐9903, MXB, China) and DAB kit (ZL1‐9019, ZSGB‐BIO, China), respectively. Counterstain was performed with hematoxylin for two minutes. Three clinical pathologists assessed the intensity of the immunostaining on each section independently in a blinded manner. At least 10 fields per specimen were surveyed.