Rabbit anti vegf c

Mice were transcardially perfused with 20 ml PBS followed by 20 ml 4% PFA. Brains were then harvested and post-fixed in 4% PFA overnight at 4 °C. Next, brains were transferred to 30% sucrose for 48 h and embedded in OCT. Sections were cut at a thickness of 50 μm and stored in 30% glycerin prior to imaging.
Sections were rinsed 3 times in PBS and blocked and permeabilized with 5% normal donkey serum and 2% Triton X-100 in PBS for 1 h at room temperature. Sections were then incubated with the following antibodies at 4 °C overnight: chicken anti-GFP (Abcam, 1:1000), rabbit Iba-1 (NovusBio, 1:100), and rabbit anti-VEGF-C (NovusBio, 1:100). The following day, respective secondary antibodies were added for 2 h at room temperature, sections were mounted using Dapi mounting media, and images were taken using a fluorescent microscope (Leica).

VEGF-C secretion in culture media was detected using a VEGF-C ELISA (NovusBio) following the manufacturer’s protocol. In brief, NSC, NSC + EC, and EC media were plated at a density of 100,000 cells/ml in 12 well plates in NSC media under baseline or GD. Media was collected at 4 and 24 h for both conditions. Following protocol, absorbance was immediately measured at 450 nm.
For immunostaining of VEGF-C and VEGFR3, NSC, NSC + EC, and EC were cultured on laminin coated coverslips for 24 h. Next, cells either remained at baseline or were glucose deprived for 4 h. The cells were fixed with 4% PFA, blocked and permeabilized, and stained overnight with rat anti-VEGFR3 (Invitrogen, 1:100) or rabbit anti-VEGF-C (NovusBio, 1:100), and phalloidin. The following day, respective secondary antibodies were added, and cells were imaged using fluorescent microscopy (Leica). VEGFR3 expression was also quantified using flow cytometry. Following 4 and 24 h of GD, NSC, NSC from NSC + EC, and EC were collected, where NSC in co-culture were detached using Accutase, ensuring EC remained adherent to the culture well. The cells were fixed with 4% PFA, blocked and permeabilized, and stained overnight. The following day, after secondary incubation, flow cytometry was conducted against VEGFR3 (NovusBio, 1:100 and analysis was done using FlowJo.