This article discusses the detection of cerebral microhemorrhages (CMHs) in rat brain tissue. CMHs are neuroinflammatory brain bleeds resulting from blood-brain barrier disruption, leading to the leakage of red blood cells into brain tissue.
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.
After isolating the brain, immerse it in 20% sucrose in PBS for at least six hours or until the brain sinks to the bottom of the tube. Change the solution to 30% sucrose, and fix it for another six hours.
Finally, prepare 10-millimeter thick brain tissue sections using a cryostat. Begin H&E staining by washing the slides in distilled water. Then, immerse in hematoxylin solution for eight minutes. Following staining, wash in running tap water for five minutes. Differentiate in 1% acid-alcohol for 30 seconds.
After washing in running tap water for one minute, stain in 0.2% ammonia water or saturated lithium carbonate solution for 30 seconds to one minute. Next, wash in running tap water for five minutes. Counterstain with eosin solution for 30 seconds to one minute. Dehydrate through 90% alcohol, then 95% alcohol, and absolute alcohol for 0.5 to 2 minutes each.
After dehydration, clear in xylene for 30 seconds. After mounting, analyze the H&E staining using a bright-field fluorescence microscope. The red blood cells released from blood vessels, which are components of the CMHs, appear in red-orange under H&E staining.
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