Expansion Microscopy Application for Calcium Protein Clustering Imaging in Cells and Brain Tissues

Author:

Rakovskaya Anastasiya1,Chigriai Margarita1,Bezprozvanny Ilya12,Pchitskaya Ekaterina1

Affiliation:

1. Laboratory of Molecular Neurodegeneration Peter the Great St. Petersburg Polytechnic University St. Petersburg Russia

2. Department of Physiology UT Southwestern Medical Center at Dallas Dallas Texas

Abstract

AbstractMany biological studies require high‐resolution imaging and subsequent analysis of cell organelles and molecules. Some membrane proteins form tight clusters, and this process is directly linked to their function. In most studies, these small protein clusters have been investigated by total internal reflection fluorescence (TIRF) microscopy, which enables imaging with high spatial resolution within 100 nm of the membrane surface. Recently developed expansion microscopy (ExM) makes it possible to achieve nanometer resolution using a conventional fluorescence microscope by physically expanding the sample. In this article, we describe implementation of ExM for imaging of protein clusters formed by the endoplasmic reticulum (ER) calcium sensor protein STIM1. This protein translocates during ER store depletion and forms clusters that support contact with plasma membrane (PM) calcium‐channel proteins. ER calcium channels such as the type 1 inositol triphosphate receptor (IP3R) also form clusters, but their investigation by TIRF microscopy is impossible due to the large distance from the PM. In this article, we demonstrate how to investigate IP3R clustering using ExM in hippocampal brain tissues. We compare IP3R clustering in the CA1 area of the hippocampus of wild‐type and 5xFAD Alzheimer's disease model mice. To facilitate future applications, we describe experimental protocols and image processing guidelines for application of ExM to membrane and ER protein clustering studies in cultured cells and brain tissues. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Expansion microscopy application for protein cluster visualization in cellsAlternate Protocol: Expansion microscopy application for protein cluster visualization in brain tissuesBasic Protocol 2: Protein cluster analysis of expansion microscopy images using ImageJ and Icy software

Publisher

Wiley

Subject

Medical Laboratory Technology,Health Informatics,General Pharmacology, Toxicology and Pharmaceutics,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3