Blocking reagent

Cells were grown on coverslips and placed on ice for 10 min prior to adding antibody (TAK-264 10 μg/mL) in cold culture medium. Cells were maintained on ice for 20 min to allow cell surface binding by the antibody. For internalization, antibody-containing medium was replaced with fresh culture medium and cells were incubated at 37°C for 3 h. Control plates were kept on ice for the same length of time. Cells were briefly rinsed in PBS and fixed for 5 min in 4% paraformaldehyde at room temperature. Cells were subsequently washed in PBS, permeabilized for 15 min in 0.5% Triton X-100, and incubated in 0.5% blocking reagent (Roche, cat. #1095176) in 100 mM Tris-HCl pH 7.5, 150 mM NaCl. An appropriate dilution of Alexa Fluor^® 488 conjugated secondary antibody (ThermoFisher Scientific) was made in 0.5% blocking reagent and added to the cells for 30 min at room temperature. Cells were then washed three times for 5 min in PBS. Coverslips were mounted on glass slides using Vecta Shield Mounting Medium (Vector Laboratories). Staining was visualized by laser scanning confocal microscopy (Zeiss Pascal) and analyzed using Axiovert software.

Nitrocellulose membranes were spotted with 5 µL of each coating mAb (0.45 mg/mL in PBS). To block additional protein-binding sites, the membrane was incubated overnight at 4° C with PBST containing 5 % blocking reagent (BioRad) with shaking. The membrane was cut to separate the original mAb spots. Each of the resulting nitrocellulose discs were then separated in one of 12 wells of a cell culture plate (Costar). Each well was filled with 500 µL of the specific polysaccharide diluted to 25 µg/mL in PBST/3 % blocking reagent. The plate was incubated for 2 h at RT with mild shaking. The specific biotinylated monoclonal antibody diluted 1:100 in 500 µL PBST/3 % blocking reagent, was added to each well followed by incubation at RT for 1 h with shaking. After extensive washing, the plate was incubated for 45 min with 500 µL of horseradish peroxidase-conjugated streptavidin (Thermo Scientific) diluted 1:5000 in PBST/3 % blocking reagent. The blot was developed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) following manufacturer instructions.

Mouse liver tissue samples were homogenized in tissue lysis buffer (25 mM Tris (pH 7.4), 2 mM Na₃VO₄, 10 mM NaF, 10 mM Na₄P₂O₇, 1 mM EGTA, 1 mM EDTA, and 1% NP-40) containing phosphatase and protease inhibitor cocktails in the OMNI BeadRuptor 24 (Omni-Inc, Kennesaw, GA). Protein was quantified using a BCA Assay (ThermoFisher Scientific, Rockford, IL) and then protein loading samples were resolved in SDS-PAGE under reducing conditions and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in blocking reagent (LI-COR, Lincoln, NE) assay system for 1 hour at room temperature and incubated with primary antibodies overnight at 4° C as follows: IDE (1:1000) from ThermoFisher Scientific (Waltham, MA) and GAPDH (1:3000) and β-actin (1:3000) from Cell Signaling Technology (Danvers, MA). Membranes were washed with tris-based saline-tween 20 (TBS-T) and the appropriate secondary antibody (1:20000, ThermoScientific, Rockford, IL) was added in blocking reagent for 1 hour at room temperature. Membranes were washed three times with TBS-T and developed using a chemiluminescence assay system. Bands on the membrane were visualized on autoradiography film. Western blot images were scanned, saved as Tiff files, inverted, and integrated density was analyzed using ImageJ software (NIH). Phosphorylated protein levels were normalized to the respective total protein levels.

Eyes tissues were fixed in 4% paraformaldehyde solution and were proceed with dehydration and embedding according to common methods by Human Biobank in National Cheng Kung University Hospital. Paraffin-embedded tissue sections (4 μm) were deparaffinized and rehydrated and subjected to heat-induced antigen retrieval by citrate buffer (pH 6.4) for 95 °C 30 min. Nonspecific binding was blocked by treatment with blocking reagent (Thermo Fisher Scientific, Cat# 003118). The sections were incubated with anti-IL-20 antibody (7E) or anti-MUC5AC antibody (Abcam, Cat# ab3649) at 4 °C overnight. 7E were prepared as previously described [37 (link), 38 (link)]. The next day, the sections were washed with PBS and incubated with the secondary antibody for 1 h. The reaction was detected using AEC chromogen stain (ScyTek Laboratories, Cat# ACG500), and the nuclei were counterstained with hematoxylin (ScyTek Laboratories, Cat# HMM500). Pictures were taken under an inverted fluorescence microscope IX71 (Olympus).