α rabbit igg hrp

Immunoblots were performed as previously described [6 (link),72 (link)]. Briefly, cells were harvested at the times indicated in lithium dodecyl sulfate (LDS) sample buffer followed by incubation at 95°C for 10 min. Denatured protein samples were run on NuPAGE 4–12% bis-Tris gels (Invitrogen). Proteins were transferred from the gel to a nitrocellulose or PVDF membrane. Membranes were blocked for 1 h in LI-COR blocking solution or in a solution of 5% (wt/vol) nonfat milk in PBS containing 0.1% Tween 20 (PBST). Membranes were incubated with primary antibody overnight at 4°C followed by 3x washes with PBST. Washed membranes were incubated in secondary antibody at room temperature for 1 h (IRDye 800CW Goat anti-Mouse IgG and IRDye 800CW Goat anti-Rabbit IgG for LI-COR; α-mouse IgG⁺HRP and α-Rabbit IgG⁺HRP, Cell Signaling 7076s and 7074s, respectively for film). Membranes were imaged with a LI-COR Odyssey imager (Lincoln, NE) or were incubated with SuperSignal West Pico or Femto chemiluminescent substrate (Thermo Scientific) and imaged by film or with a chemiluminescence imager.

Cells were harvested at times indicated in lithium dodecyl sulfate (LDS) sample buffer and incubated at 95°C for 10 min. Protein samples were run on NuPAGE 4–12% bis-Tris gels (Invitrogen). Proteins were transferred from the gel to nitrocellulose or PVDF membranes for imaging via LI-COR Odyssey imager (Lincoln, NE) or film, respectively. Membranes were blocked in either LI-COR blocking solution or 5% powdered milk-PBS containing 0.1% Tween 20 (PBST) for 1 hr. After blocking, membranes were incubated with primary antibody overnight at 4°C. Membranes were washed 3 times with PBST, incubated in secondary antibody for 1 hr at room temperature (IRDye 800CW Goat anti-Mouse IgG and IRDye 800CW Goat anti-Rabbit IgG for LI-COR; α-mouse IgG⁺HRP and α-Rabbit IgG⁺HRP, Cell Signaling 7076 s and 7074 s, respectively for film). Membranes were washed 3 times in PBST. Nitrocellulose membranes were imaged with a LI-COR Odyssey imager. PVDF membranes were incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and exposed to film.

Whole-mount Prox1 immunofluorescence was performed as previously described (Koltowska et al., 2015 (link); Shin et al., 2017 (link)) in Tg[fli1a:nEGFP]^y7. Antibodies used were chicken α-GFP (1:400, Abcam, ab13970) rabbit α-Prox1 (1:500, AngioBio Co, 11-002P), α-rabbit IgG-HRP (1:1000, Cell Signaling Technology, BioNordika, 7076S) and TSA-Cy3 (1:50, PerkinElmer, Akoya, NEL744001KT). The trunks of zebrafish were cut at the junction of the yolk and the yolk extension and flat mounted in clearing solution Omnipaque™ (350 mg I/ml iohexol, GE Healthcare) on a glass-bottom imaging dish. The images were taken with the inverted confocal Leica SP8 DSL system using a 25× water objective.
Prox1 and nEGFP double-positive cells were identified in the PCV and secondary sprouts. Neighbouring cells were defined by adjacent nEGFP+ EC, normally corresponding to a cell in the PCV and the other in the secondary sprout. When two neighbouring EC expressed Prox1, the occurrence was quantified. The number of incidences was counted per seven somites in every embryo, in three biological replicates.