Biotinylated secondary antibody goat anti mouse

For immunostaining analyses, fixed EFPs were dehydrated, embedded in paraffin and cut into 8-μm thick sections. These sections were deparaffinized in xylene and ethanol, subjected to antigen retrieval by heating the samples at 95 °C for 20 min in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in Tris-buffered saline (TBS) for 30 min. After blocking the tissue samples with 1% bovine serum albumin and 0.025% Triton X-100 for 2 h, sections were stained with primary antibodies against α-smooth muscle actin (α-SMA; 1:500; Sigma-Aldrich) at 4 °C overnight. Sections were then incubated with biotinylated secondary antibody goat anti-mouse (1:500; Abcam) for 1 h at RT. Following a 10 min incubation of streptavidin-peroxidase (Abcam), samples were exposed to DAB solution (Abcam) for 3 min and rinsed thoroughly with water. Specimens were counterstained with a 1/10 hematoxylin dilution for 1 min and mounted for imaging with a Nikon Eclipse E600 microscope (Nikon Instruments Inc.). The tissue area surrounding the hydrogel was used to quantify positively stained vessels, lumen area and thickness of vessels. Sections of five different fat pads of each condition were analyzed.