Donkey anti goat conjugated to fitc

Our first step, in vivo, was to establish that EPCs from the construct had the ability to migrate from the Construct into the myocardium. In order to assess EPC migration, eGFP⁺ EPCs from transgenic rats were utilized to create the Construct (20 mg/ml Fibrin, 17 × 10⁶ EPCs/ml). This Construct was then implanted onto ischemic myocardium following LAD ligation. The hearts were explanted 1 week following implant, flushed with PBS, and distended with OCT embedding compound (Electron Microscopy Sciences). Visualization was performed in the peri-infarct borderzone, which was defined as one microscopic field from the infarct. Hearts treated with Construct were compared to IC. Sections were briefly washed, then fixed in 4% paraformaldehyde and then stained for anti-GFP and anti-α smooth muscle actin (SMA, pericytes). Primary antibodies used for indirect immunofluorescence were goat anti-GFP (1:200, Rockland) and rabbit anti-SMA (1:150, Abcam). Secondary antibodies included donkey anti-goat conjugated to FITC (1:200, Abcam), and donkey anti-rabbit conjugated to Alexa Fluor 594 (1:200, Abcam). Nuclei were counterstained with DAPI (Vector Laboratories). Construct was compared to Control (isolated LAD ligation) and IC.

Sections were briefly washed three times in PBS, fixed in 4% paraformaldehyde for 10 minutes at room temperature, and blocked in 10% Fetal Bovine Serum (FBS) (Gibco) for 1 hour at 37°C. Primary antibodies were diluted 1:150 in PBS and incubated for 2 hours at 37°C. Secondary antibodies were diluted in PBS and incubated for 1 hour at 37°C. Primary antibodies included sheep anti-vWF (endothelial cells of vasculature) conjugated to FITC (Abcam) and rabbit anti-SMA (Abcam). Secondary antibodies (1:200) were donkey anti-goat conjugated to FITC (Abcam), and donkey anti-rabbit conjugated to Alexa Fluor 594 (Abcam). Nuclei were stained with DAPI (Vector Laboratories). Vasculature was quantified using ImageJ (NIH). Measurements were made 1 high power field from the infarct at 20× magnification.