The superiority and feasibility of 2,3,5-triphenyltetrazolium chloride-stained brain tissues for molecular biology
Yajuan Wang1, Qingrong Zhang1, Shuchi Zhang2
1Department of Anesthesiology, Huashan Hospital, Fudan University, Shanghai, China.
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.
Insights
2,3,5-triphenyltetrazolium chloride (TTC) staining is reliable for analyzing brain tissue after stroke. TTC-stained tissue allows for precise molecular analysis in specific regions, including microglia expression, without degradation.
Area of Science:
- Neuroscience
- Molecular Biology
- Pathology
Background:
- 2,3,5-triphenyltetrazolium chloride (TTC) staining is standard for assessing cerebral infarct volume in stroke models.
- Microglia morphology varies regionally after ischemic stroke, necessitating region-specific molecular analysis.
- This study investigates the utility of TTC-stained brain tissue for detailed molecular profiling.
Purpose of the Study:
- To evaluate the feasibility and necessity of using TTC-stained brain tissue for molecular biology experiments.
- To compare molecular analysis of TTC-stained tissue with traditional methods.
- To confirm the suitability of TTC-stained tissue for regional analysis based on microglia characteristics.
Main Methods:
- Comparison of brain tissue processed with an improved TTC staining method versus traditional penumbra sampling.
- Analysis using real-time quantitative PCR (RT-PCR), Western blot, and immunofluorescence.
- Assessment of protein and RNA integrity in TTC-stained samples.
Main Results:
- No significant protein or RNA degradation was observed in TTC-stained brain tissue.
- TREM2, a microglial marker, showed significant differences between groups in the penumbra region.
- The improved TTC staining method proved feasible and necessary for accurate regional analysis.
Conclusions:
- TTC-stained brain tissue is suitable for molecular biology experiments without limitations.
- Precise regional positioning of TTC-stained tissue offers significant advantages for molecular analysis.
- This method enhances the study of stroke pathophysiology by enabling detailed regional molecular characterization.
