Technical review: Colocalization of antibodies using confocal microscopy

Abstract

Imaging of fluorescent signals is often performed for investigating localization of proteins, organelles, ions, and cells. Whether two molecules of interest are located in the same area, (i.e., colocalization) is an issue faced by researchers. Conventional colocalization is where two or more antigens are viewed in the same section by secondary antibodies labeled with fluorescent compounds. Conventional colocalization only shows the coexistence of molecules without any quantification. This method does not examine whether the intensity of staining for two or more molecules varies in synchrony, which would be expected if they were part of the same complex. Various methods have been developed to solve these issues and are discussed in this article. This article discusses techniques and issues of colocalization associated with confocal microscopy. In particular, the article discusses methods used for quantification of colocalization, such as intensity correlation analysis, an automated method based on spatial statistics, and fuzzy system models. Anat Rec, 297:183-187, 2014.

Attenuated succinate accumulation relieves neuronal injury induced by hypoxia in neonatal mice

“…2G, H ). Moreover, the most positive LC3B signals overlapped with TOMM20, which is the mitochondrial marker [ 39 ] (Fig. 2A–F ).…”

Single molecule fluorescence spectroscopy for quantitative biological applications

“…Once target molecules can be identified by their single‐molecule fluorescence, it is straightforward to look at their locations and to measure the distance between molecules. Conventional colocalization use the fluorescence signals to identify whether two molecules of interest are located in the same area [67, 68]. However, the spatial resolution of the optical microscopy is on the order of a few hundred nanometers, which is much larger than the size of most biological molecules.…”

EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues

“…Another major issue in cardiovascular pathology is that the high-precision and high-throughput analysis of microcirculation demands a magnification exceeding values obtainable by means of light microscopy (LM, 400-fold) and an image acquisition rate superior to confocal microscopy (which is commonly limited to 630-fold magnification, being additionally confounded by antigen expression features and background staining) ( 7 , 8 ). Transmission electron microscopy provides a perfect magnification range, but the sample preparation is technically challenging, is inevitably associated with a tremendous reduction of the sample amount, and does not permit the representative serial sectioning of the vessels ( 9 ).…”