Immunofluorescence Microscopy

Abstract

Visualizing fluorescence‐tagged molecules is a powerful strategy that can reveal the complex dynamics of the cell. One robust and broadly applicable method is immunofluorescence microscopy, in which a fluorescence‐labeled antibody binds the molecule of interest and then the location of the antibody is determined by fluorescence microscopy. The effective application of this technique includes several considerations, such as the nature of the antigen, specificity of the antibody, permeabilization and fixation of the specimen, and fluorescence imaging of the cell. Although each protocol will require fine‐tuning depending on the cell type, antibody, and antigen, there are steps common to nearly all applications. This article provides protocols for staining the cytoskeleton and organelles in two very different kinds of cells: flat, adherent fibroblasts and thick, free‐swimming Tetrahymena cells. Additional protocols enable visualization with widefield, laser scanning confocal, and eSRRF super‐resolution fluorescence microscopy. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Immunofluorescence staining of adherent cells such as fibroblastsBasic Protocol 2: Immunofluorescence of suspension cells such as TetrahymenaBasic Protocol 3: Visualizing samples with a widefield fluorescence microscopeAlternate Protocol 1: Staining suspension cells adhered to poly‐l‐lysine‐coated coverslipsAlternate Protocol 2: Visualizing samples with a laser scanning confocal microscopeAlternate Protocol 3: Generating super‐resolution images with SRRF microscopy