TTC (2, 3, 5-triphenyltetrazolium chloride) is a fat-soluble light-sensitive compound. TTC staining has been used to determine the infarct extent induced by ischemia in tissues. TTC is substrate of dehydrogenase as the proton receptor of pyridine-nucleotide enzyme of respiratory chain. It act with dehydrogenase of tissue to generate red compounds. The ischemia tissues have low dehydrogenase activity that cannot react with TTC and left unstained.
TTC staining solution
1. Isolate tissue sample
The experimental animals are anaesthetized. Isolate tissues (e.g. brain, heart) quickly, place in -20℃ to quick-freeze for 20-30 min. The tissue samples can be preserved at -80℃ for a short time if it is not urgent.
2. Prepare frozen section
Let the frozen tissue bulk unfreeze slightly. Section the tissue bulk into serial 2-3 mm thick slices with Leica manual slice.
3. TTC staining
Immerse slides in preheating TTC staining solution, incubate for 15-30 min at 37℃ avoiding light. Gently shake the slides every 5-8 min to make staining solution contact with slides uniformly.
4. Photograph and analyze result
Stop the staining reaction by adding fixing solution and preserve slides in dark condition. Take pictures for slides the next day.
Normal tissues show red while the infarct areas show sickly white.
Infarct area shows pale white, and normal area is red.
1. Use fresh samples without fix. Slices should be thin and sharp. Control the quick-freeze time properly to facilitate slicing. Keep slices flat.
2. The stained slides are valid in several months when preserved in fixing solution and avoid light. Slides may fade in sunlight.
3. TTC staining solution is valid in one month when kept in 4 ℃ and dark condition. Ensure to restore to normal room temperature before use.
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