Introduction to the principle of TTC staining

TTC staining is a reaction between TTC and succinate dehydrogenase in the mitochondria of living cells, which produces a red crescent, which is used to express the viability of the cells. In 37 degree condition, it is a common index to evaluate cerebral ischemia injury.

TTC staining principle
TTC (2,3,5-triphenyltetrazolium chloride) is a fat-soluble photosensitive complex. It was first synthesized in 1894 to detect the viability of seeds, and began to be used to detect ischemic infarction in mammalian tissues in 1958. It is the proton receptor of the pyridine-nucleoside structure enzyme system in the respiratory chain. It reacts with the dehydrogenase in the normal tissue, but the dehydrogenase activity in the ischemic tissue decreases and can not react, so it will not change pale.

Ttc can be reduced by enzymes with reductive activity in living cells, such as succinate dehydrogenase, to form pink reductase products, so the living tissue is pink, and the activity of reductase in dead cells has disappeared. As a result, ttc cannot be restored, so the dead tissue is white.

For seed or plant tissue, the staining result is that living tissue is stained with different degrees of red, dead tissue or lifeless tissue is not stained. For ischemic infarction tissues, the tissue necrosis dehydrogenase activity loss was pale, but the normal tissue was dark red. The commonly used staining concentration of TTC was 2% (w / v), which could also be adjusted according to the tissue type.

TTC dyeing step
1, brain extraction: the brain can be taken directly after anesthesia or perfused with normal saline. Because brain tissue is not fixed with paraformaldehyde, so softer, brain should be taken more carefully, to maintain the integrity of the brain.

And 2, freezing in a refrigerator for 20 minutes in a 20-degree refrigerator to be convenient for slicing.

3. Slice: cut into 5-6 pieces, and cut one piece every 2 mm. The first knife is at the midpoint of the pre-brain pole and the visual cross-connecting line, the second knife is at the sight-crossing part, the third knife is at the part of the funnel handle, and the fourth knife is arranged between the funnel handle and the rear leaf tail pole. If the area is to be calculated, it should be cut evenly or with a special brain groove.

4. The slices were placed in the TTC with a conventional concentration of 2% and also 1% or less.

5. Cover with the foil paper, put it into the 37 C temperature box for 15-30min, turn the brain slices from time to time, and make the uniform contact to the dyeing liquid.

6, the effect after staining: subcortical infarction.

TTC dye is a kind of lipid-soluble photosensitive complex which can be used to detect the viability of seeds and the ischemic infarction of mammalian tissues. The detection mechanism is that TTC itself can act as an indicator of redox activity. Dehydrogenase in living cells (especially succinate dehydrogenase in mitochondria) can reduce TTC to TPF (1,3,5 triphenylformazan).

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