This study investigates the integrity of the blood-brain barrier (BBB) following restricted blood flow in a rat model. The methodology involves the use of fluorophore-tagged albumin to assess BBB damage and subsequent electron microscopy for detailed analysis.
Take a rat subjected to restricted blood flow to the right brain hemisphere, resulting in BBB damage.
Intravenously inject fluorophore-tagged albumin, which crosses the damaged BBB, reaching the brain.
Obtain chemically-fixed brain sections.
Add blocking buffer to prevent non-specific antibody binding.
Apply peroxidase-conjugated antibodies targeting the fluorophore-tagged albumin.
Add a chromogenic substrate, which the peroxidase converts into an electron-dense precipitate.
Introduce osmium tetroxide to fix membrane lipids and enhance contrast.
Dehydrate the sections with increasing alcohol concentrations.
Add uranyl acetate, which binds to nucleic acids and proteins to increase contrast.
Dehydrate again with increasing alcohol concentrations, finishing with propylene oxide for resin embedding. Embed the sections in resin.
Microscopically identify and excise the chromogen-stained regions.
Re-embed these sections in resin and prepare ultrathin slices.
Mount the slices on electron microscopy (EM) grids.
Stain with lead citrate to enhance extracellular matrix contrast.
Proceed with EM to examine BBB integrity.
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