TY - JOUR
T1 - Efficient isolation of brain capillary from a single frozen mouse brain for protein expression analysis
AU - Ogata, Seiryo
AU - Ito, Shingo
AU - Masuda, Takeshi
AU - Ohtsuki, Sumio
N1 - Funding Information:
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: We are grateful for partial financial support in the form of Grant-in-Aid for Scientific Research(B), Mishima Kaiun Memorial Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, and JST CREST Grant Number JP171024167, Japan.
Publisher Copyright:
© The Author(s) 2020.
PY - 2021/5
Y1 - 2021/5
N2 - Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood–brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis. We successfully isolated brain capillaries from a single frozen mouse brain by using a bead homogenizer in the brain homogenization step and combination of cell strainers and glass beads in the purification step. Western blot and proteomic analysis showed that proteins expressed at the BBB in mouse brain capillaries isolated by the developed method were more enriched than those isolated from a pool of five mouse brains by the standard method. By using the developed method, we further verified the changes in expression of BBB proteins in Glut1-deficient mouse. The developed method is useful for the analysis of various mice models with low numbers and enables us to understand, in more detail, the physiology and pathology of BBB.
AB - Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood–brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis. We successfully isolated brain capillaries from a single frozen mouse brain by using a bead homogenizer in the brain homogenization step and combination of cell strainers and glass beads in the purification step. Western blot and proteomic analysis showed that proteins expressed at the BBB in mouse brain capillaries isolated by the developed method were more enriched than those isolated from a pool of five mouse brains by the standard method. By using the developed method, we further verified the changes in expression of BBB proteins in Glut1-deficient mouse. The developed method is useful for the analysis of various mice models with low numbers and enables us to understand, in more detail, the physiology and pathology of BBB.
KW - Blood–brain barrier
KW - GLUT1 deficiency syndrome
KW - absolute protein expression
KW - brain capillary isolation
KW - quantitative protein expression profile
UR - http://www.scopus.com/inward/record.url?scp=85088595575&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85088595575&partnerID=8YFLogxK
U2 - 10.1177/0271678X20941449
DO - 10.1177/0271678X20941449
M3 - Article
C2 - 32703112
AN - SCOPUS:85088595575
SN - 0271-678X
VL - 41
SP - 1026
EP - 1038
JO - Journal of Cerebral Blood Flow and Metabolism
JF - Journal of Cerebral Blood Flow and Metabolism
IS - 5
ER -