Quantifying Cerebral Microhemorrhage Characteristics

Right hemispheres were fixed in 4% paraformaldehyde (Boston BioProducts, Ashland, MA) at 4 °C, examined for surface CMH, and sectioned into 40-μm coronal sections using a vibratome (Technical Products International, Inc., St. Louis, MO). Every fourth, fifth, sixth, and seventh section was collected. Every sixth section was stained with H&E by Research Services Core offered by the Department of Pathology and Laboratory Medicine at UCI Medical Center to detect fresh (acute) CMH. Every seventh section was stained with Prussian blue (PB) to detect hemosiderin (a marker of sub-acute CMH) [16 (link)]. Briefly, sections were stained using 5% potassium hexacyanoferrate trihydrate (Sigma, St. Louis, MO) and 5% hydrochloric acid (Sigma, St. Louis, MO) for 30 min, rinsed in water and counterstained with Nuclear Fast Red (Sigma, St. Louis, MO), dehydrated, and cover slipped. Remaining sections were used for immunohistochemistry. CMH were counted at a × 20 magnification by a blinded observer, and digitized images were used to determine CMH size (μm²) and positive area (expressed as a percent of total area analyzed), by an observer blinded to the experiment using RGB CMH analyzer program and NIH Image J software 1.62, respectively [16 (link)].