Biotin conjugated isolectin b4

For detection of ChAT, GLUT-4 and cardiac troponin I (cTnI, a cardiac marker), seven micrometer thick sections were probed with anti-ChAT (Abcam, AB181023), anti-GLUT-4 (NovusBio, NBP1-49533) and biotin-conjugated anti-cTnI antibodies (NovusBio, NB110-2546B) in a sequential manner.
For microvascular analysis, seven micrometer thick sections were probed with biotin-conjugated Isolectin-B4 (Vector laboratories, B1205; 1:200) and anti-α-smooth muscle actin conjugated with Cy3™ (Sigma-Aldrich, C6198; 1:800) to detect endothelial cells and smooth muscle cells, respectively. The vascular density was expressed as the mean number of Isolectin⁺ cells (for capillaries) or αSMA⁺ and Isolectin⁺ cells (for arterioles) per mm² of cardiac tissue.
To determine the level of fibrosis, sections were stained with 0.1% Direct Red 80 (Sigma Aldrich, 3,665,548)/Picric acid solution (Sigma Aldrich, 197,378). The fibrotic area was normalized to total tissue area and expressed as fold change relative to the control group.

For histological analysis, tissue sections collected from mouse heart were fixed overnight in freshly prepared 4% paraformaldehyde, followed by cryoprotection in 30% sucrose in PBS solution. The sections were then embedded in Tissue-Tek OCT compound and frozen by placing them on cold isopentane/2-methylbutane. Frozen sections were stored at −80°C until cryosectioned. Seven-micron-thick myocardial cryosections were then probed with biotin-conjugated Isolectin-B4 (1:200 dilution, Vector laboratories, USA) and anti-α-smooth muscle actin conjugated with Cy3 (1:800 dilution, Sigma-Aldrich, USA) to detect endothelial cells (to determine the capillary density) and smooth muscle cells (to determine the arteriole density), respectively. DAPI (1:1,000 dilution, Santa Cruz Biotechnologies, USA) was used to stain the nuclei. Images were captured at 200× magnification using a confocal microscope (Nikon A1). The density of capillaries was expressed as the mean number of isolectin⁺ cells/mm² of cardiac tissue. The density of arterioles (<50 μm lumen size) was expressed as the mean number of αSMA⁺isolectin⁺ cells/mm² of cardiac tissue.