TY - JOUR
T1 - Can we use 2,3,5-triphenyltetrazolium chloride-stained brain slices for other purposes? The application of western blotting
AU - Sanchez-Bezanilla, Sonia
AU - Nilsson, Michael
AU - Walker, Frederick R.
AU - Ong, Lin Kooi
N1 - Funding Information:
We would like to acknowledge the insights and comments provided by Rebecca Hood on early versions of the manuscript. Funding. This study was supported by the Hunter Medical Research Institute (HMRI 896), Faculty of Health and Medicine Pilot Grant, the Priority Research Centre for Stroke and Brain Injury Research Support Grant, the Mary Costello Alzheimer?s Pilot Grant, and The University of Newcastle, Australia. LKO and SS-B also acknowledge the support from the Research Advantage for ECR Higher Degree by Research (HDR) Scholarship and Greaves Family Postgraduate Scholarships in Medical Research (HMRI 1054).
Funding Information:
This study was supported by the Hunter Medical Research Institute (HMRI 896), Faculty of Health and Medicine Pilot Grant, the Priority Research Centre for Stroke and Brain Injury Research Support Grant, the Mary Costello Alzheimer’s Pilot Grant, and The University of Newcastle, Australia. LKO and SS-B also acknowledge the support from the Research Advantage for ECR Higher Degree by Research (HDR) Scholarship and Greaves
Publisher Copyright:
© Copyright © 2019 Sanchez-Bezanilla, Nilsson, Walker and Ong.
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2019/7/30
Y1 - 2019/7/30
N2 - 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.
AB - 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.
KW - 2
KW - 3
KW - 5-triphenyltetrazolium chloride
KW - hippocampus
KW - infarct
KW - stroke
KW - thalamus
KW - western blotting
UR - http://www.scopus.com/inward/record.url?scp=85072203653&partnerID=8YFLogxK
U2 - 10.3389/fnmol.2019.00181
DO - 10.3389/fnmol.2019.00181
M3 - Article
C2 - 31417355
AN - SCOPUS:85072203653
VL - 12
JO - Frontiers in Molecular Neuroscience
JF - Frontiers in Molecular Neuroscience
SN - 1662-5099
M1 - 181
ER -