Abstract
2,3,5-Triphenyltetrazolium chloride (TTC) staining is a commonly used method to determine the volume of the cerebral infarction in experimental stroke models. The TTC staining protocol is considered to interfere with downstream analyses, and it is unclear whether TTC-stained brain samples can be used for biochemistry analyses. However, there is evidence indicating that, with proper optimization and handling, TTC-stained brains may remain viable for protein analyses. In the present study, we aimed to rigorously assess whether TTC can reliably be used for western blotting of various markers. In this study, brain samples obtained from C57BL/6 male mice were treated with TTC (TTC+) or left untreated (TTC−) at 1 week after photothrombotic occlusion or sham surgery. Brain regions were dissected into infarct, thalamus, and hippocampus, and proteins were extracted by using radioimmunoprecipitation assay buffer. Protein levels of apoptosis, autophagy, neuronal, glial, vascular, and neurodegenerative-related markers were analyzed by western blotting. Our results showed that TTC+ brains display similar relative changes in most of the markers compared with TTC− brains. In addition, we validated that these analyses can be performed in the infarct as well as other brain regions such as the thalamus and hippocampus. Our findings demonstrate that TTC+ brains are reliable for protein analyses using western blotting. Widespread adoption of this approach will be key to lowering the number of animals used while maximizing data.
Highlights
Damaged/dead tissue remains white showing the absence of living cells, and thereby indicating the infarct region (Bederson et al, 1986; Li et al, 1997)
We found no significant differences in neither the thalamus nor hippocampus (Figure 2A)
Protein levels of Neuronal nuclei (NeuN) were significantly reduced in stroke mice in the infarct and thalamus areas of both TTC− (NeuN infarct p < 0.0001, NeuN thalamus p = 0.0021) and TTC+ (NeuN infarct p < 0.0001; NeuN thalamus p = 0.0086) group
Summary
Damaged/dead tissue remains white showing the absence of living cells, and thereby indicating the infarct region (Bederson et al, 1986; Li et al, 1997). Western blotting is a common method used to detect and analyze protein levels This technique involves the separation of denatured proteins based on their molecular weight and visualization using antibodies specific to the target protein (Burnette, 1981; Liu et al, 2014). This study only analyzed the protein expression profile of the metalloprotease-disintegrin ADAM12 and the housekeeping protein β-actin Additional work in this field has demonstrated that TTC-stained brain sections can be used for immunohistochemical quantification of Collagen IV and immunofluorescence analyses (Li L. et al, 2018; Li Z. et al, 2018). Taken into account previous data, the main objective of the present study was to investigate whether TTC-treated tissues can be processed for downstream biochemical analyses, by western blotting. We hypothesized that TTC treatment would not interfere with protein quantification in the regions studied
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