Optimal cutting temperature compound oct

Infarct size measurement: Excised hearts were sectioned axially into 1 mm thickness (McIlwain 800 Series, Vibratome, St Louis, MO) and stained with 2,3,5-triphenyltetrazolium chloride (TTC, from GFS Chemicals, Powell, OH) following the manufacturer's protocol. TTC images were acquired on a flatbed scanner (Hewlett Packard, Palo Alto, CA), and microsphere images were acquired on the IVIS spectrum. Image analysis was performed in ImageJ, with infarction size across the heart slices normalized to the area-at-risk (AAR).
Immunohistochemistry: Excised hearts were embedded in Optimal Cutting Temperature compound (OCT, from Thermo Fisher), and 10 μm short-axis cryo-sections were collected. Direct immunohistochemistry of CD68 was performed with anti-CD68 antibody conjugated to Alexa Fluor 647 fluorophore (Santa Cruz Biotechnology, Dallas, TX) per manufacturer's instructions. Diamidino-2-phenylindole (DAPI, from Thermo Fisher) staining was performed to visualize the nuclei. Microscopy was performed on a TISSUEFAXS system (TissueGnostics GmbH, Vienna, Austria), with a 40X objective lens (Zeiss), DAPI (for DAPI), GFP (for Gd-TO), and Alexa 647 (for CD68) filter settings. Images were acquired on a 12 bit CCD camera (Pixelink, Ottawa, Ontario, Canada), with the TissueFAXS image acquisition software, and analyzed in Image J (NIH).