Vegfr3

6 month-old mouse brains in an AKR background were sectioned and paraffin-embedded [21 (link)]. Sections on slides were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pre-treatment with a pressure cooker for 10 min in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked in 0.3% H₂O₂ in methanol for 10 min and non-specific binding with 10% dried milk solution. For this specific experiment, tissue sections were incubated with primary antibodies against human proteins that cross-react with mouse; PROX-1 (1:400; Acris), or VEGFR3 (1:40; R&D Systems) for 1 h at RT, followed by biotinylated anti-rabbit IgG (1:200; Dako) or biotinylated anti-mouse IgG (1:200; Dako) for 30 min at RT and Avidin–Biotin complex (30 min; Dako). Colour was developed with di-aminobenzidine/H₂O₂ [32 (link)].
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.

IHC for CTGF (sc-14939; Santa Cruz Biotechnology Inc., Dallas, TX), VEGF-C (Zymed Laboratories, South San Francisco, CA), D2-40 (BioLegend, San Diego, CA), LYVE-1 (Acris Antibodies GmbH, Herfold, Germany), and VEGFR-3 (R&D Systems) was performed as previously reported^{13 (link),34 (link),53 (link)}. To determine positive areas on stained sections, 10 random fields per section were chosen and photographed. LYVE-1-positive lymphatic vessels were identified and quantitated using Image J software, and the density was calculated for the analysis of lymphangiogenesis. CTGF and VEGF-C expression were semiquantitatively classified as follows: 0, absent; 1, mild; 2, moderate; 3, extensive^{13 (link),54 (link)}.
ISH to detect CTGF mRNA was performed on formalin-fixed paraffin-embedded rat diaphragm samples according to previously described methods^{18 (link),55 (link)}.

Formalin-fixed, paraffin-embedded tissue Sections (8 µm) from the frontal and occipital cortices were cut from cases 1–6 listed in Table 1. Sections were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pre-treatment with a pressure cooker for 10 min in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked in 0.3% H₂O₂ in methanol for 10 min and non-specific binding with 10% dried milk solution. Tissue sections were incubated with primary antibodies; MRC1 (1:1000; R&D systems), PROX1 (1:400; Acris), LYVE1 (1:50; Abcam), LYVE1(1:200; R&D Systems), PDPN (1:350; Sigma-Aldrich), and VEGFR3 (1:40; R&D Systems) for 1 h at RT, followed by biotinylated anti-rabbit IgG (1:200; Dako) or biotinylated anti-mouse IgG (1:200; Dako) for 30 min at RT and Avidin–Biotin complex (30 min; Dako). Colour was developed with di-aminobenzidine/H₂O₂ [32 (link)].
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.

HLECs were grown on coverslips and treated with or without TCDCA or TCA (100 µM) for 24 h. The cells were fixed with 4% paraformaldehyde, permeabilized with ice-cold methanol, and blocked with goat serum. The cells were incubated with total primary YAP (Novus Biologicals, Littleton, CO, USA) or β-Catenin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies for 2 h at room temperature. The cells were washed and then incubated with fluorescence-tagged secondary antibodies for 1 h at room temperature. After washing, the coverslips were mounted on slides and imaged with 40X objectives in a fluorescent microscope (Olympus). Liver and liver nodes were isolated from control (FVB) and Mdr2^−/− mice and embedded in the OCT (optimal cutting temperature) (TissueTek, Torrance, CA, USA) compound. The tissue sections from the liver and liver lymph node from the control FVB and Mdr2^−/− mice were cut at a thickness of 10 µm with a Leica CM 1860 (Leica, Heidelberg, Germany) cryostat machine at a core facility of the Department of Medical Physiology at the College of Medicine, Texas A&M University (College Station, TX, USA). These sections were stained with CK19 (MyBioSource, San Diego, CA, USA), LYVE-1 (R&D Systems, Minneapolis, MN, USA), and VEGFR3 (R&D Systems, Minneapolis, MN, USA) in 1:100 dilution.

Colon sections were fixed using 4% paraformaldehyde and embedded in paraffin. The paraffin block was cut into 4 μm sections, deparaffinized with xylene and rehydrated with ethanol. After blocking for 1 h, the colon tissue was incubated overnight at 4 °C with anti-mouse lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (Angiobio, Del Mar, CA, USA), anti-vascular endothelial growth factor receptor-3 (VEGFR-3) (R & D systems, Minneapolis, MN, USA), and anti-ER-HR3 antibodies (BMA, Augst, Switzerland). The sections were treated with AEC substrate-chromogen (DakoCytomation, Glostrup, Denmark) to visualize the immunocomplexes. Immunohistochemical staining was visualized under a Nikon Eclipse 80i light microscope (Nikon Instruments Inc., Melville, NY, USA). The densities of LYVE-1-positive and VEGFR-3-positive areas were measured in 12 randomly selected fields at a magnification of ×400 using the ImageJ software.