Immunofluorescence Staining

Abstract

This unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to a fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescence microscope. This unit describes the widely used and powerful technique of localization of proteins in cells by immunofluorescence. The location can be determined by double labeling with an antibody directed against a protein of known location. The technique can be used as a supplement to immunolocalization by electron microscopy and subcellular fractionation. It allows not only identification of the antigen distribution in the cell but also a survey of the dynamic aspects of protein movements in the cell—on and off membranes, into and out of the nucleus, and through membrane traffic pathways. © 2015 by John Wiley & Sons, Inc.

Three‐Dimensional Patterning of the ECM Microenvironment Using Magnetic Nanoparticle Self Assembly

“…no. H3570) Confocal microscope Additional reagents and equipment for immunofluorescence staining (UNIT 4.1; Donaldson, 2015) and confocal microscopy (UNIT 4.5; Smith, 2001) Fix the sample 1. Gently aspirate the culture medium from the LabTek coverglass chambers containing the cells grown in the 3D anisotropic matrix, wash the sample twice with 200 μl PBS, and remove the wash buffer.…”

Nuclei Isolation Staining (NIS) Method for Imaging Chromatin‐Associated Proteins in Difficult Cell Types

“…Immunostaining is a widely used technique to characterize protein localization patterns in intact cells. The general approach includes fixation of intact cells and permeabilization using detergent to facilitate antibody penetration (see Current Protocols article: Donaldson, 2015). The efficiency of this approach can vary among different cell types.…”

“…For chromatin-associated proteins, immunostaining is conventionally applied to whole cells. A permeabilization step using detergent is typically included to facilitate antibody penetration towards the interior of the nucleus (see Current Protocols article: Donaldson, 2015). Although this approach can be used to image chromatin-associated proteins in many cell types, it remains challenging for a subset of difficult cell types.…”

Mobile Quantitative Colocalization Analysis of Fluorescence Microscopy Images

“…Mobile quantitative colocalization analysis (QCA) assumes understanding of fluorescence microscopy [UNIT 4.2 (Herman, 2002) and UNIT 4.5 (Smith, 2001)] and immunostaining techniques (UNIT 4.3;Donaldson, 2015). In addition, since the workflow for quantification of colocalization on mobile computers replicates the workflow on desktops, it is also recommended to be familiar with our protocol on the desktop platform (UNIT 4.19;Zinchuk & Grossbacher-Zinchuk, 2014).…”