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

After removing the brain, including the brain stem, from the skull, place the brain, with its ventral side up, in the brain matrix. Depending on the weight of the animals, choose the correct size of the brain stainless-steel matrix. Using blades, restrict the frontal and caudal parts of the brain. Then, put the blades partially into the channels between the first and the last blades.

When all the blades are inserted and arranged in parallel, press all the blades down with the palm at the same time to cut the brain into 2-millimeter coronal slices. Then, grasp the blades firmly along the sides with two fingers, and remove them together with the sliced brain from the matrix. Arrange the brain slices one by one in a tray, ensuring that the anterior surface of each slice is always facing up.

Next, pour warm 1% triphenyl tetrazolium chloride solution and PBS onto the brain slices, and incubate for 8 minutes at 37 degrees Celsius in the dark. After incubation, transfer the brain slices into a blue plastic tray to capture images. Arrange the brain slices in sequential order from the frontal to the caudal part, and use a scalpel to separate the hemispheres in the sagittal plane.