Alexa fluor conjugated goat anti rabbit secondary antibody

The M0 macrophages cultured in 6-well plates were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 20 min, and then blocked with BSA at room temperature for 30 min. Thereafter, samples were incubated with rabbit anti-rat STAT3 primary antibody (1 : 1000, Proteintech Group, Inc., Wuhan, China) overnight at 4°C. After 3 washes with PBST, cells were incubated with Alexa Fluor-conjugated goat anti-rabbit secondary antibody (1 : 1000, Invitrogen) at room temperature for 1 h, avoiding light. After further washing with PBST, nuclei were stained with 4′,6-diamidino-2-phenylindole for 5 min, in the dark. The fluorescent signals were analyzed using an inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Immunofluorescence and phase-contrast images were used for analyzing the lymphatic vessels. For immunofluorescence staining, a macrofluidic device after experiments was fixed with 4% paraformaldehyde and incubated for 20 min at room temperature. Fixed cells were permeabilized using 0.1% Triton X-100 for 10 min. To prevent nonspecific binding, the samples were blocked with 1% BSA and incubated for 1 h at room temperature. Primary antibodies with 1% BSA, anti–VE-cadherin, anti-laminin, and anti-VEGFR3 (all from Abcam, United Kingdom) were filled into the channels and incubated for 2 h. After washing with 1x PBS three times, a mixture of Alexa Fluor conjugated goat anti-rabbit secondary antibody, rhodamine-phalloidin, and 4′,6-diamidino-2-phenylindole (all from Thermo Fisher Scientific) was filled in the device and incubated for 2 h. Images were obtained using a fluorescence microscope and a confocal microscope (LSM700; Carl Zeiss). A morphological analysis was conducted using ImageJ.