TTC Staining of Rat Brain Tissue Slices: A Staining Procedure to Differentiate Viable and Necrotic Tissue in Tissue Slices After Ischemia-Reperfusion Injury

During brain ischemia, impaired blood flow to the brain reduces oxygen and nutrient supply to the tissue, causing brain damage. After brief ischemia, as the blood flow to the brain gets restored, reperfusion with increased oxygen supply results in reactive ion species overproduction, leading to oxidative tissue damage, known as ischemia-reperfusion injury.

To assess brain damage following ischemia-reperfusion injury in a rat model, first, harvest the rat brain. Rinse in a chilled buffer to remove debris and blood. Section the brain to obtain coronal slices of desired thicknesses. Carefully transfer the slices into a buffer-containing tray - with anterior surfaces facing up - for subsequent staining.

Add solution of warm triphenyl tetrazolium chloride, TTC, a water-soluble redox dye, over the brain slices. Incubate under dark conditions, as TTC is light-sensitive. TTC enters cells within the tissue section.

In living cells, active mitochondrial succinate dehydrogenase enzyme in the presence of cofactors reduces TTC, colorless when oxidized, into water-insoluble, red formazan, staining viable cells deep red. However, necrotic cells lack dehydrogenase activity; thus, TTC remains in oxidized form, staining the cells white.

Post-incubation, transfer the brain slices into an imaging tray. Separate the hemispheres in the sagittal plane. Image the brain slices.

Healthy tissue appears red, while damaged tissue appears white, differentiating necrotic tissue from viable tissue and enabling estimation of the extent of tissue damage.