Recombinant Alexa Fluor® 647 Anti-ERG antibody [EPR3864] (ab196149)

For mouse retina immunostaining, eyes were collected at the indicated time points and fixed in 4% PFA in PBS for 1 h at room temperature (RT). After two PBS washes, retinas were micro-dissected and stained. Briefly, retinas were blocked and permeabilized with 0.3% Triton X-100, 3% fetal bovine serum (FBS) and 3% donkey serum in PBS. Samples were then washed twice in PBLEC buffer (1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂ and 1% Triton X-100 in PBS). Biotinylated isolectinB4 or primary antibodies (Panel a, ab196149 1/100 dilution) were diluted in PBLEC buffer and tissues were incubated in this solution for 2 h at RT or overnight at 4 °C. After five washes in blocking solution diluted 1:2, samples were incubated for 1 h at RT with Alexa-conjugated secondary antibody. After two washes in PBS, retinas were mounted with Fluoromount-G. To detect EdU-labeled DNA, an additional step was performed before mounting using the Click-It EdU kit.

Dll4/Notch signalling inhibition induces context-dependent proliferative effects. a–d Confocal micrographs of the postnatal retinal vasculature from animals treated at P5 with IgG (control) or anti-Dll4 for 24 h (a, b) or 48 h (c, d). Anti-Erg (red) labels EC nuclei. EdU labels the nuclei of all cells in S-phase in the previous 4 h. Blue nuclei mark non-endothelial cells in S-phase, and double-positive (Erg+/EdU+) cell nuclei are pseudocoloured green to better highlight ECs in S-phase. Scale bars, 200 μm.

RLCs selectively differentiate to intraglomerular mesangial cells during EC model.
Panel A shown only. Representative confocal microscopy images for day 0 and day 7 of β-gal and the EC markers ERG (upper panels) or CD31 (lower panels) co-stained kidney slices.

Immunostainings and Acid fuchsin orange G (AFOG) staining were performed on paraffin-embedded 4-μm kidney sections. Antibodies were diluted in 1% BSA/TBS. Primary antibodies were incubated overnight at 4°C, secondary antibodies and Avidin D-conjugated horseradish peroxidase were incubated for 2 hours at room temperature. All immunofluorescence samples were counterstained with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI) and mounted with mowiol mounting medium. Endogenous peroxidase activity was suppressed with 3% hydrogen peroxide solution. Samples were counterstained with hematoxylin, dehydrated and mounted. The following antibodies were used: WT-1 (ab202639), nephrin, PDGFRβ (ab91066), α8-integrin, CD31, ERG (ab196149), β-galactosidase (ab9361), renin, CD45 (ab64100).

Mt4-mmp expression during early mouse embryonic development.

Panel K shown only: Double-labeled cells for the endothelial markers ERG (red, arrows in K) and β-gal (green) demonstrate that cells expressing Mt4-mmp in this location are endothelial cells. Abbreviations: da, dorsal aorta; fp, floor plate; s, somite; NCC, neural crest cells; NT, neural tube. Scale bars: 30 μm (J-L).

For immunohistochemical procedures, embryos were fixed in 4% PFA in PBS 0.1M pH 7.2 by immersion or perfusion depending on the developmental stage, cryoprotected in a 30% sucrose solution, and then embedded in OCT and sectioned in the cryostat at 20 μm thickness in the transverse plane. Immunohistochemistry was performed following standard protocols. Primary antibodies used include: polyclonal anti-CD31 hamster (1/1000), anti-β-galactosidase rabbit (1/1000; ab4761, Abcam), anti-FoxA2 mouse (1/250), anti-Nkx6.1 mouse (1/1000), anti-Olig2 rabbit (1/1000), anti-ERG-647 rabbit (1/500; ab196149, Abcam) and anti-WT-1 mouse (Wilms Tumor-1; 1/50;). Sections were incubated with the primary antibody diluted in PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA), for 48 h at 4°C. Subsequently, the sections were rinsed in PBS and incubated for 2 hours at room temperature with 488 or 594-Alexa™-conjugated fluorescent antibodies (1/1000). Sections were counterstained with Hoechst (1:1000) for 5 min at room temperature to visualize nuclei.

ab196149 staining ERG in THP-1 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab196149 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).