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
Incorporation of a stable‐isotope metabolic tracer into cells or tissue can be followed at submicron resolution by multi‐isotope imaging mass spectrometry (MIMS), a form of imaging secondary ion microscopy optimized for accurate isotope ratio measurement from microvolumes of sample (as small as ∼30 nm across). In a metabolic MIMS experiment, a cell or animal is metabolically labeled with a tracer containing a stable isotope. Relative accumulation of the heavy isotope in the fixed sample is then measured as an increase over its natural abundance by MIMS. MIMS has been used to measure protein turnover in single organelles, track cellular division
- NSF-PAR ID:
- 10175118
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Cell Biology
- Volume:
- 88
- Issue:
- 1
- ISSN:
- 1934-2500
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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 asTetrahymena Basic 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 -
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This article was corrected on 25 July 2022. See the end of the full text for details.
Basic Protocol 1 : Genetic engineering strategy for the generation of modular light‐activated protein dimerization unitsSupport Protocol 1 : Molecular cloningBasic Protocol 2 : Cell culture and transfectionSupport Protocol 2 : Production of dark containers for optogenetic samplesBasic Protocol 3 : Confocal microscopy and light‐dependent activation of the dimerization systemAlternate Protocol 1 : Protein recruitment to intracellular compartmentsAlternate Protocol 2 : Induction of organelles’ membrane tetheringAlternate Protocol 3 : Optogenetic reconstitution of protein functionBasic Protocol 4 : Image analysisSupport Protocol 3 : Analysis of apparent on‐ and off‐kineticsSupport Protocol 4 : Analysis of changes in organelle overlap over time -
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