The superiority and feasibility of 2,3,5-triphenyltetrazolium chloride–stained brain tissues for molecular biology experiments based on microglial properties

Abstract

Background: TTC (2,3,5-triphenyltetrazolium chloride) staining is the most commonly used method in identifying and assessing cerebral infarct volumes in the transient middle cerebral artery occlusion model. Given that microglia exhibit different morphologies in different regions after ischemic stroke, we demonstrate the superiority and necessity of using TTC-stained brain tissue to analyze the expression of various proteins or genes in different regions based on microglia character. Methods: We compared brain tissue (left for 10 min on ice) from the improved TTC staining method with penumbra from the traditional sampling method. We identified the feasibility and necessity of the improved staining method using real time (RT)-PCR, Western blot, and immunofluorescence analysis. Results: There was no protein and RNA degradation in the TTC-stained brain tissue group. However, the TREM2 specifically expressed on the microglia showed a significant difference between two groups in the penumbra region. Conclusions: TTC-stained brain tissue can be used for molecular biology experiments without any restrictions. In addition, TTC-stained brain tissue shows greater superiority due to its precise positioning.