Af349

Collected cells were lysed in SDS buffer and sonicated on ice. After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific, Waltham, MA, USA), proteins were resolved by SDS-PAGE and transferred to PVDF membrane by electroblot analysis. Membranes were blocked in Tris-buffered saline containing 5% skim milk and 0.05% Tween-20 and incubated with the following primary antibodies: goat anti-VEGFR3 (1:1000) (AF349) (R&D systems) and rabbit anti-β actin (1:8000) (Medical and Biological Laboratories, Nagoya, Aichi, Japan). Horseradish peroxidase conjugated anti-goat, anti-rabbit IgG (1:4000) were used as secondary antibodies for chemiluminescence detection. Signals were obtained by enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA). The bands were analyzed by densitometry using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).

Paraffin-embedded specimens were deparaffinized with xylene and ethanol, and then antigen-activated with a 10 mM citrate buffer (pH 6.0) in a microwave oven. Next, 1% bovine serum albumin (BSA) (Wako, Osaka, Japan) diluted with PBS was added dropwise to prevent nonspecific protein binding, and the mixture was placed in a humid box for 30 min. Next, goat anti-VEGF-C (AF752) (1:50) (R&D Systems, Minneapolis, MN, USA) diluted with PBS was added dropwise as a primary antibody, and the mixture was reacted overnight in a humid box at 4 °C. Then, a secondary antibody reaction was performed for 30 min at room temperature using Alexa Fluor 488 or 546 (1:400) (Life Technologies, Carlsbad, CA, USA). For immunocytochemistry of trabecular meshwork cells, cells were seeded into a six-well plate with cover glass and fixed with 4% paraformaldehyde for 15 min and permeabilized by 0.1% Triton X-100 for 10 min. Cells were incubated in 5% BSA/PBS for 30 min and then incubated with a primary goat antibody against VEGFR3 (1:50) (AF349) (R&D systems) at 4 °C overnight prior to the exposure to Alexa Fluor 546 anti-goat IgG (1:400) for 1 h at room temperature. Nuclear staining was performed with 4′,6-diamino-2-phenylindole (DAPI) (1:500) (Lonza, Basel, Switzerland).
The observation of the section was performed with a fluorescence microscope Keyence BZ-9000 series (BIOREVO, Keyence, Osaka, Japan).