Ab150372

MAGE-A1 expression was thoroughly examined in LUAD cell lines and tissue samples. For the qPCR, the sequences of the primers are listed in Additional file 7: Table S2. For the western blotting analysis, two types of primary monoclonal antibodies were obtained from Abcam (ab193330, ab243935, Abcam, Cambridge, MA, USA). The protocols of the qPCR test and western blotting analysis were described previously [24 (link), 25 (link)]. The immunofluorescence test was conducted following the protocols described in our previous study [26 (link)]. Cells were incubated with FITC-labeled human anti-MAGE-A1 antibody (Abcam, ab212590) in the dark. 4′-6-diamidino-2-phenylindole (DAPI, Biotium, Hayward, CA) was used for nuclear staining. The ubiquitous Desmoglein 2 (DSG-2) was employed as a positive control (Abcam, ab150372). Immunohistochemistry (IHC) analysis was performed as previously described [27 (link), 28 (link)]. TMA sections were incubated with mouse monoclonal anti-MAGE-A1 antibody (Abcam, ab193330). The secondary antibody used was horseradish peroxidase-conjugated anti-mouse antibody. Phosphate-buffered saline (PBS) was used as a negative control.

After fixation with 4% PFA, the cells were permeabilized using 0.1% Triton X‐100 (5 min, RT) and were incubated with rabbit anti‐DSG2 antibodies (Abcam, Cambridge, UK, #ab150372, 1 : 100) for 1 h at RT. Afterward, the cells were washed several times with PBS and were incubated with secondary anti‐rabbit IgG antibodies conjugated with Alexa 647 (Abcam, #ab150075, 1 : 100). After several additional washing steps with PBS, the cells were embedded in Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and were analyzed using confocal microscopy. F‐actin was costained with phalloidin conjugated to Texas Red according to the manufacturer's instructions (Thermo Fisher Scientific, #T7471).

For IHC, 4-µm paraffin-embedded sections were baked at 60 °C for 1 h, deparaffinized with xylene, rehydrated with a series of graded alcohols, and microwaved in EDTA antigen retrieval buffer. Then, the sections were blocked with 10% goat serum before incubation with a primary antibody at 4 °C overnight, followed by HRP-conjugated secondary antibody incubation for 30 min at room temperature. DAB was added to detect antibody binding. Once brown color appeared, the sections were immersed in distilled water to stop the reaction. The sections were counterstained with hematoxylin, dehydrated in graded alcohols and mounted. The primary antibodies were rabbit anti-human DSG2 monoclonal antibody (ab150372, Abcam, Britain) and mouse anti-human D2–40 monoclonal antibody (MAB-0567, MXB, China). The DSG2 staining results were scored based on the following criteria: (i) percentage of positive tumor cells in the tumor tissue: 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–70%) and 4 (71–100%); and (ii) staining intensity: 0 (none), 1 (weak), 2 (moderate), and 3 (strong). The staining index was calculated as the staining intensity score × the proportion of positive tumor cells (range from 0 to 12). The staining score of 6 was defined as the cutoff. Thus, patients with different positive staining levels of DSG2 expression were divided into low- and high-staining groups.

A validation cohort was created using 30 samples of pancreatic cancer and associated paracancerous tissues that had been validated by normal pathological investigation from our pathology department. All samples have comprehensive clinical information. Following serial sectioning of the tissue wax blocks, sample sections were collected and kept in a 4 °C freezer for later use. We subsequently performed IHC assays on formalin-fixed de-paraffinized sections using DSG2 (Abcam, ab150372), LDHA (Abcam, ab52488), and RACGAP1 (Abcam, ab134972) antibodies and took images with microscopy as described previously^{21 (link)}.

To identify immune cell populations from 2-week Dsg2^−/− hearts, we employed the chromogenic tissue-staining method using the DAB detection system. The automated Ventana Classic and XT system (Roche Diagnostics, UK) was used to process our paraffin-embedded heart samples. The antibodies used were rat monoclonal CD45 (Ab25386, Abcam, UK) and rat monoclonal F4/80 (MCA497GA Clone CI:A3-1, Serotec, UK). The OmniMap anti-rabbit HRP kit (760–4311, Roche Diagnostics, UK) was used to detect the antibodies of interest. The samples were counterstained with haematoxylin; slides were sealed with mountant and coverslip and allowed to dry. Sections from Dsg2^+/+ and Dsg2^−/− hearts were also stained with Dsg2 antibody (Ab150372, Abcam, UK) to confirm that Dsg2 had been deleted in the heart. Slides from the various histological stains were scanned with a Pannoramic 250 high-throughput scanner (HistoTech, Budapest, Hungary), and representative images from 2-week and 10-week samples with scale bars were processed with the Panoramic Viewer software (HistoTech, Budapest, Hungary).