Detection of Cerebral Microhemorrhages in a Rat Model

Cerebral microhemorrhages, or CMHs, are neuroinflammatory brain bleeds caused by blood-brain barrier disruption, resulting in the leakage of red blood cells, or RBCs, into brain tissue.           

To detect CMHs, begin with fixed brain tissue from a rat with CMHs.

Immerse the tissue in sucrose for cryoprotection.

Using a cryo-microtome, section the tissue and collect a section onto a glass slide.

Wash to remove residual fixative.

Apply hematoxylin dye bound to a metal cation that interacts with negatively charged DNA, staining the nucleus reddish-purple.

Follow with acid-alcohol to remove excess dye, then apply an alkaline bluing agent to neutralize the acidic environment and convert the dye to its blue form, enhancing contrast.

Wash the tissue with alcohol to improve stain penetration, then apply eosin to stain the RBCs red-orange and tissue components pink.         

Dehydrate the tissue using increasing alcohol concentrations and clear it with xylene for microscopic visualization.          

Capture images to detect the red-orange colored leaked RBCs, confirming CMHs.