Goat anti mouse cy2

To detect the intercellular adherence junction protein VE-cadherin, ECs on HFM were gently washed with Dulbecco’s Phosphate-Buffered Saline (DPBS) and fixed in 4% (v/v) paraformaldehyde for 15 min, at room temperature (RT). Following rinsing with Phosphate-Buffered Saline (PBS; w/o Ca²⁺/Mg²⁺), ECs were permeabilized and blocked for unspecific antibody binding by incubation with 0.25% Triton-X100, diluted in Tris-buffered saline supplemented with 5% (serum of the respective host of the secondary antibody) for 20 min at RT. Then, the samples were rinsed three times with PBS and incubated with primary anti-VE-cadherin antibody (diluted at 1:50 in 1% bovine serum albumin (BSA) in PBS w/o Ca²⁺/Mg²⁺; AbD Serotec, Puchheim, Germany), for 1 h at RT. After three washing steps, fluorescence-labeled secondary antibodies, goat anti-mouse Cy2 (Jackson ImmunoResearch, Cambridge, UK) were applied for 1 h at RT in the dark. Nuclei were stained with Höchst3334 dye (10 µg/mL) added to the secondary antibody incubation for the last 20 min. Samples incubated with isotype-matching antibodies were prepared as controls in parallel. After embedding in mounting medium (Immumount, Dako, Germany), microscopic images were obtained using the above-mentioned fluorescence microscope.

After HSD2-immunolabeling, forebrain sections from EB-treated rats (n = 6) were processed to visualize catecholaminergic neurons and fibers in the hypothalamus at the level of the VMH by immunolabeling for tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis. Sections were incubated in the primary antibody (Millipore; mouse anti-TH; MM_NF-MAB318)³ diluted 1:5000 in 2% NGS for 1 h at room temperature, and then for approximately 48 h at 4°C. On the second day of processing, tissue was brought to room temperature for 1 h, rinsed in 2% NGS, and then incubated for approximately 5 h in the secondary antibody (Jackson Immunoresearch; goat anti-mouse Cy2) diluted 1:300 in 2% NGS.