Dako protein block

Tissue sections were deparaffinized in xylene followed by decreasing grades of ethanol. Samples were then quenched in 3% hydrogen peroxide and pretreated to promote antigen retrieval by using a steaming method with citrate buffer solution (pH 6.0). Slides were incubated in Dako Protein Block (Agilent), and then incubated with primary antibody to KRT18 (Abcam, ab181597) at a dilution of 1:2000 or Ki67 (Cell Signaling Technology, 12202S) at a dilution of 1:200. KRT18 antibody was validated using mouse liver as positive control, and Ki67 antibody was validated using mouse intestine as positive control. The samples were washed and incubated in Dako Rabbit Polymer (Agilent). Slides were further incubated with chromogen diaminobenzidine tetrahydrochloride (DAB). For amplification and visualization of our KRT18 or Ki67 signal, slides were counterstained with hematoxylin, rehydrated, and then mounted. For duct quantification, slides were imaged on an EVOS FL Auto Imaging System (ThermoFisher Scientific) and analyzed using ImageJ (NIH). All KRT18 or Ki67 positively stained ducts were detected using the Color Threshold tool in ImageJ. Based on KRT18 IHC images, ducts were quantified and sorted into two categories, either large or small ducts as defined by a cutoff of 1906 µm². This cutoff was derived from taking the arithmetic mean of all ducts detected in the vehicle-treated group.

Blood was collected in heparin-containing vials when mice were killed, centrifuged at 4°C (3,000 rpm in 20 min), and stored at −80°C. Plasma Gal-3 levels were detected by a mice Galectin-3 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA) in twin duplicates wells, following protocols provided by the manufacturer. The detection range of the plasma Gal-3 was 8.19–2,000 pg/mL, and the limit of detection was 6 pg/mL.
Paraffin-embedded LV sections (6 μm) were prepared and used for Gal-3 immunofluorescent staining within mice. For Gal-3 immunofluorescent staining, after dewaxed, heat-induced antigen retrieval and permeabilization were carried out (with 10 mM of Na-citrate buffer containing 0.05% Tween 20; pH 6.0; 95°C for 25 min) followed by blocking with DAKO Protein Block (X0909, Agilent, 1 h at room temperature). Sections were incubated with primary goat anti-mouse Gal-3 (1:100, AF1197, R&D Systems) overnight at 4°C, then they were incubated with the secondary antibody, Alexa Fluor 594 donkey anti-goat IgG (1:200, A11058, Invitrogen by Thermo Fisher Scientific). The cardiomyocyte boundary was revealed by wheat-germ-agglutinin FITC staining (1:80, FL-1021, Vector Labs, 1 h at room temperature). Images were acquired with an Olympus BX61 fluorescent microscope.

For immunostaining, the sections were fixed in buffered 4% Paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature. They were then blocked for one hour in DAKO protein block (Agilent Technologies, Santa Clara, CA, USA) to reduce unspecific binding. For labelling endothelial cells, the sections were stained overnight at 4 °C d with goat anti-CD31 (Abcam, Cambridge, UK) for detecting endothelial cells, rat anti-CD68 (Bio-Rad Laboratories, Hercules, CA, USA) for macrophage detection and with anti-GFP (Abcam) for detecting GFP cells. They were subsequently incubated with AlexaFluor conjugated secondary antibodies (Thermo Fisher Scientific) for one hour at 37 °C. After washing, the sections were mounted on cover slips with DAKO mounting medium containing DAPI (Agilent Technologies). Fluorescence images were obtained using a Zeiss ELYRA PS.1 LSM 780 confocal imaging system (Carl Zeiss AG).

De-identified human liver specimens from liver explants were obtained from the Liver Center Tissue Bank at the University of Kansas Medical Center. All studies using human tissue samples were approved by the Human Subjects Committee of the University of Kansas Medical Center.
Immunohistochemistry were performed as previously described [18 (link)]. After deparaffinization and rehydration, antigen retrieval was achieved by heating in a pressure cooker for 5 min in 10 mM of sodium citrate (pH 6). Peroxidase activity was blocked by incubation in 3% H₂O₂ for 10 min. Sections were rinsed three time in PBS/PBS-T (0.1% Tween-20) and incubated in Dako Protein Block (Dako, Agilent Technologies, Santa Clara, CA) for 10 min. After removal of blocking solution, slides were placed into a humidified chamber and incubated overnight with primary antibodies in blocking buffer (3% normal goat serum in PBS) and incubated over night at 4 °C. After washing, slides were covered with SignalStain Boost IHC Detection Reagent (Cell Signaling Technologies, Boston, MA) for 30 min at room temperature. After washing two times with PBS-T, the Substrate-Chromgen Solution (VECTOR NovaRED, Substrate Kit, Vector Laboratories, Burlingame, CA) was applied, slides were incubated 5–10 min and counterstained with Hemtoxylin. Images were acquired using a Nikon Eclipse 80i microscope (Nikon Americas Inc., Melville, NY).

Immunohistochemistry was performed as previously described (Li et al., 2018 (link); Zhao et al., 2019 (link)). After deparaffinization and rehydration, antigen retrieval was achieved by heating in a pressure cooker for 5 min in 10 mM of sodium citrate (pH6). Peroxidase activity was blocked by incubation in 3% H₂O₂ for 10 min. Sections were rinsed three time in PBS/PBS-T (0.1% Tween-20) and incubated in Dako Protein Block (Dako, Agilent Technologies, Santa Clara, CA) for 10 min. After removal of blocking solution, slides were placed into a humidified chamber and incubated overnight at 4°C with primary antibodies in blocking buffer (4% normal goat serum in PBS). After washing, slides were covered with SignalStain Boost IHC Detection Reagent (Cell Signaling Technologies, Boston, MA) for 30 min at room temperature. After washing two times with PBS-T, the substrate-chromgen solution (VECTOR NovaRED, Substrate Kit, Vector Laboratories, Burlingame, CA) was applied, and the slides were incubated for 5–10 min and counterstained with hemtoxylin. Images were acquired using a Zeiss Axiolab 5 Digital Lab Microscope (Carl Zeiss AG, Jena, Germany).

Lung tissue sections were fixed as reported above. Lung samples underwent antigen retrieval (pH 9 buffer) using a Dako PT Link pre-treatment module (Agilent). Samples were washed and blocked for 10 min (Dako protein block; Agilent, Santa Clara, CA) before being treated with primary antibodies overnight. Mouse anti-COL1A1, ARG1, and rabbit anti-FDPS, CD206 (Abcam, Cambridge, United Kingdom) antibodies were used. Alexa Fluor 488-conjugated goat/anti-mouse and Alexa Fluor 647 goat/anti-rabbit (Invitrogen, Waltham, CA, United States) were used as secondary antibodies. Glass cover slips were placed onto slides and mounted with DAPI-containing fluoroshield (Abcam). Microscopy was performed on a Widefield Epifluorescence Ti2 microscope equipped with a Nikon DS-Qi2 camera and fluorescence was quantified using ImageJ software.