Ab109185

After washed with PBS, cells were lysed with RIPA lysate (Beyotime, Shanghai, China). Protein concentrations were determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Protein was resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membrane (Invitrogen, USA). Afterwards, membranes were incubated with primary antibodies (Anti‐E‐Cadherin, ab1416, 1:50; Anti‐Mucin‐1, ab109185, 1:1000; Anti‐Vimentin, ab8978, 1/100; Anti‐Fibronectin, ab23750, 1 μg/mL; Anti‐c‐Myc, ab39688, 1/500; Anti‐β‐Actin, ab11003, 1/500, Abcam, Cambridge, MA, USA; Anti‐MAPK1, 1:100, Bosterbio, USA). Subsequently, we added IgG‐HRP labelled goat anti‐mouse secondary antibody (ab205719, 1:10000, Abcam, Cambridge, MA, USA). The immunoreactive proteins were visualized using the ECL Detection System (Life technologies, Gaithersburg, MD, USA).

MEECs were grown to an appropriate density and fixed in 4% (w/v) paraformaldehyde for 15 min, permeabilized with 0.5% Triton-X-100 for 15 min, and blocked with 5% bovine serum albumin for one hour at room temperature. After blocking, cells were labeled with the primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, rabbit anti-CD44, Proteintech, 15675-1-AP; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321), and the secondary antibodies (1:100, fluorescently labeled goat anti-mouse lgG-cy3, BA1031, Boster company, Wuhan, China) according to the manufacture’s protocol. DAPI (0.5 mg/ml, D3571, Thermo Fisher) was used to stain the nucleus. Then, the fluorescence was detected by Leica DM4000B fluorescence microscope.

The expression levels of Arg-1 and GPC-3 in 237 resected ICC samples were evaluated by immunohistochemistry (IHC). All specimens were fixed in 4% neutral formaldehyde solution, dehydrated with gradient alcohol, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E). H&E-stained tissues were observed under a light microscope (Nikon, 80i, Japan). ICC tissue sections were also stained with the following antibodies: rabbit monoclonal anti-Arg-1 ab109185.html">antibody (SP15; Jianlun Biology Technology, China), mouse monoclonal anti-GPC-3 ab109185.html">antibody (ab129381; Abcam, USA), and rabbit monoclonal anti-MUC1 ab109185.html">antibody (ab109185; Abcam, USA).

The DAB staining was performed using a commercial DAB Detection Kit (Maixin biotech company). ALI 3D cultures or tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. The sections were deparaffinized and hydrated through xylene and graded alcohol series and rinsed for 5 min in tap water. Antigens were retrieved by heating samples in a microwave for 15 min in citric acid buffer. The primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, Chicago, IL, USA, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, Cambridge, UK, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321) were incubated, respectively, on the slides at 4 °C overnight, then detected with the reaction-amplified reagent for 20 min, and conjugated with high-sensibility enzyme conjugated lgG polymer. Reactants were visualized with the fresh-prepared DAB chromogenic solutions for 3 to 5 min. Hematoxylin somatic cell staining reagent was used to counter-stain nuclei. All the coverslips were mounted on the glass slides using anti-quenching Fluoroshield™ histology mounting medium (Sigma-Aldrich) and visualized under a fluorescence microscope (BX51TF, Olympus company, Tokyo, Japan) with magnification 40 ×.

This study used normal endometrial cells (NECs), KLE cells, RL952 cells, Ishikawa cells, and ECC-1 cells. They were maintained and cultured as previously described [15 (link)]. The cell lines used in this study were as follows: normal endometrial cells (NEC, CP-H058, Procell, Wuhan, China), KLE (CL-0133, Procell, Wuhan, China), RL952 (CL-0197, Procell, Wuhan, China), Ishikawa (CL-0283, Procell, Wuhan, China), and ECC-1 (BS-C163325, BinSuiBio, Shanghai, China).
The following antibodies were used: Anti-METTL5 (16791-1-AP, Proteintech, Rosemont, USA), anti-Ki-67 (ab15580, Abcam, Cambridge, USA), anti-CEA (ab207718, Abcam, Cambridge, USA), anti-CA199 (ab3982, Abcam, Cambridge, USA), anti-CA153 (ab109185, Abcam, Cambridge, USA), anti-HE4 (13, ab200828, Abcam, Cambridge, USA), anti-MLH1 (ab92312, Abcam, Cambridge, USA), anti-MSH2 (ab212188, Abcam, Cambridge, USA), anti-MSH6 (ab92471, Abcam, Cambridge, USA), anti-PMS2 (ab110638, Abcam, Cambridge, USA), and anti-β-actin (M01263-2, Boster, Wuhan, China).

Lung samples were fixed for 24 h with 4% paraformaldehyde in PBS, embedded in paraffin, and sectioned into 5-μm-thick slices. The slices were stained with hematoxylin and eosin, Elastica van Gieson, and periodic acid-Schiff stain, according to standard protocols.
For immunohistochemistry, tissue sections were deparaffinized, rehydrated, and rinsed with PBS several times. Antigen retrieval was performed in 10 mM citrate buffer (pH 6) at 121°C for 15 min. Slides for enzyme labeled antibody method were additionally incubated in 3% H₂O₂ for endogenous peroxidase inactivation for 30 min at room temperature, then all slides were blocked with 5% normal goat serum in PBS for 1 h, incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature, and finally mounted with 3,3ˊ-diaminobenzidine. Anti-Ki67 (ab16667, 1:1000; Abcam) and anti-MUC1 (ab109185, 1:250; Abcam) were used as primary antibodies.
Slides for bright field were imaged on a Leica DM6000 (Leica Microsystems GmbH) microscope. Immunofluorescent slides were imaged on an Invitrogen EVOS M7000 Imaging System (Thermo Fischer Scientific). For quantitative analysis, 10 high-power views at a ×400 magnification were counted for each sample (n = 3) and analyzed using ImageJ software (National Institutes of Health).

Paraffin-embedded, formalin-fixed LMS and LPS specimens were used for IHC. Sections were incubated overnight in a humidified container at 4°C with the primary antibodies of MUC1 (1:100, ab109185; Abcam) and CD11c (1:100, ab52632, Abcam). After three times washing, tissue sections were incubated with the secondary antibody conjugated with streptavidin–horseradish peroxidases for 1 h at room temperature. The slides were stained with 3, 3-diaminobenzidine tetrahydrochloride (DAB) and the nuclei were counterstained with hematoxylin. Immunostaining on each slide was assessed by experienced pathologists to examine the percentage of MUC1 or CD11c positive tumor cells and presented as histochemistry score (H-score). H-score = Σpi(i+1) where i is the intensity score and pi is the percent of the cells with that intensity.