Secondary donkey anti rabbit cy3

RDEB rat and WT skin tissue samples were frozen in optimal cutting temperature (OCT, Sakura Finetek USA, Torrance, CA) and sectioned at 6 microns on a cryostat. Sections were fixed in room temperature acetone for 5 minutes. Tissue sections were blocked with 10% normal donkey serum for 1 hour (Jackson Immunoresearch, West Grove, PA). Primary antibody C7 (1:200 LifeSpan BioSciences, Seattle, WA) was incubated overnight at 4°C. Slides were washed with 1× PBS. Secondary donkey anti-rabbit Cy3 (Jackson Immunoresearch, 1:500) was applied for 1 hour at room temperature. Slides were washed with 1× PBS and then cover-slipped with hard-set DAPI, 4,6-diamidino-2-phenylindole (Vector Labs, Burlingame, CA). Slides were examined by confocal fluorescence microscopy (Olympus BX61, Olympus Optical, Tokyo, Japan).

Pulmonary vascular remodeling was measured by degree of occlusion of capillary arteries on 7 μm-thick sections of frozen lung tissue. Slices were fixed with acetone for 10 minutes at room temperature and then saturated with human (10%) and donkey (10%) sera in PBS for 1 hour at room temperature. We used mouse anti alpha Smooth Muscle Actin (α-SMA)-FITC from Sigma-Aldrich (clone 1A4, dilution 1/100) and rabbit anti von Willebrand Factor (Dako, dilution 1/100). Primary antibodies were incubated overnight at 4°C. Antibody binding was detected with secondary donkey anti-rabbit-Cy3 (1/100) from Jackson ImmunoResearch. One lung section per rat was analyzed (n = 14 rats per group) and all capillary arteries were classified in 4 categories: not muscularized, partially muscularized, fully muscularized and completely occluded according the presence or not of SMC-actin staining around vWF + precapillary arteries (<50 μm).