Can We Use 2,3,5-Triphenyltetrazolium Chloride-Stained Brain Slices for Other Purposes? The Application of Western Blotting

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.

Growth Hormone Treatment Promotes Remote Hippocampal Plasticity after Experimental Cortical Stroke

“…Protein extraction and Western blotting were performed as previously described [ 63 , 64 ]. Hippocampus samples were sonicated in 300 μL lysis buffer (50 mM tris(hydroxymethyl)aminomethane buffer pH 7.4 containing 80 μM ammoniummolybdate, 5 mM β -glycerolphosphate, 1 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate, 1 mM sodium pyrophosphate, 1 mM sodium vanadate, 1 cOmplete™ protease inhibitor cocktail tablet, and 1 PhosSTOP™ phosphatase inhibitor cocktail tablet) and centrifuged at 14000 g for 20 min at 4 °C.…”

Similar cognitive deficits in mice and humans in the chronic phase post-stroke identified using the touchscreen-based paired-associate learning task

“…Photothrombotic occlusion was performed as described previously with minor modifications 19,25 . Briefly, mice were anaesthetized by 2% isoflurane during the surgical procedure on a temperature-regulated (37 °C ± 1) stereotaxic frame.…”

Activation of MC1R with BMS-470539 attenuates neuroinflammation via cAMP/PKA/Nurr1 pathway after neonatal hypoxic-ischemic brain injury in rats

“…Brain tissues were used for Western blot analysis after first being stained with TTC and imaged. Previous studies demonstrated that TTC-stained brain tissues could be used for quantitative gene and protein expression analyses using real-time polymerase chain reaction (RT-PCR) and Western blot in models of ischemic stroke including in the HI rat model without altering the expression of proteins [ 52 – 54 ]. After TTC staining, slices were imaged and recorded at 48 h post-HI, and the brain slices were instantly divided into the ipsilateral and contralateral cerebrums, snap frozen in liquid nitrogen, and then stored in a − 80 °C freezer for Western blot.…”