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
Visualization of gene products in
- NSF-PAR ID:
- 10304193
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 1
- Issue:
- 11
- ISSN:
- 2691-1299
- 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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Abstract Cleavage under targets and release using nuclease (CUT&RUN) is a recently developed chromatin profiling technique that uses a targeted micrococcal nuclease cleavage strategy to obtain high‐resolution binding profiles of protein factors or to map histones with specific post‐translational modifications. Due to its high sensitivity, CUT&RUN allows quality binding profiles to be obtained with only a fraction of the starting material and sequencing depth typically required for other chromatin profiling techniques such as chromatin immunoprecipitation. Although CUT&RUN has been widely adopted in multiple model systems, it has rarely been utilized in
Caenorhabditis elegans , a model system of great importance to genomic research. Cell dissociation techniques, which are required for this approach, can be challenging inC. elegans due to the toughness of the worm's cuticle and the sensitivity of the cells themselves. Here, we describe a robust CUT&RUN protocol for use inC. elegans to determine the genome‐wide localization of protein factors and specific histone marks. With a simple protocol utilizing live, uncrosslinked tissue as the starting material, performing CUT&RUN in worms has the potential to produce physiologically relevant data at a higher resolution than chromatin immunoprecipitation. This protocol involves a simple dissociation step to uniformly permeabilize worms while avoiding sample loss or cell damage, resulting in high‐quality CUT&RUN profiles with as few as 100 worms and detectable signal with as few as 10 worms. This represents a significant advancement over chromatin immunoprecipitation, which typically uses thousands or hundreds of thousands of worms for a single experiment. The protocols presented here provide a detailed description of worm growth, sample preparation, CUT&RUN workflow, library preparation for high‐throughput sequencing, and a basic overview of data analysis, making CUT&RUN simple and accessible for any worm lab. © 2022 Wiley Periodicals LLC.Basic Protocol 1 : Growth and synchronization ofC. elegans Basic Protocol 2 : Worm dissociation, sample preparation, and optimizationBasic Protocol 3 : CUT&RUN chromatin profilingAlternate Protocol : Improving CUT&RUN signal using a secondary antibodyBasic Protocol 4 : CUT&RUN library preparation for Illumina high‐throughput sequencingBasic Protocol 5 : Basic data analysis using Linux -
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Abstract This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouse peritonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll‐like receptor agonists and TNF‐α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation‐mediated maturation of interleukin (IL)‐1β and IL‐18 and pyroptosis. Since the well‐established agonists for NLRP1 are inconsistent between mice and humans, Basic Protocol 2 describes how to activate the NLRP1 inflammasome in human PBMCs. Basic Protocol 3 describes how to purify, crosslink, and detect the apoptosis‐associated speck‐like protein containing a CARD (ASC) pyroptosome. Formation of the ASC pyroptosome is a signature of inflammasome activation. A limitation of ASC pyroptosome detection is the requirement of a relatively large cell number. Alternate Protocol 1 is provided to stain ASC pyroptosomes using an anti‐ASC antibody and to measure ASC specks by fluorescence microscopy in a single cell. Intraperitoneal injection of lipopolysaccharides (LPS) and inflammasome agonists will induce peritonitis, which is seen as an elevation of IL‐1β and other proinflammatory cytokines and an infiltration of neutrophils and inflammatory monocytes. Basic Protocol 4 describes how to induce NLRP3 inflammasome activation and peritonitis by priming mice with LPS and subsequently challenging them with monosodium urate (MSU). The method for measuring cytokines in serum and through peritoneal lavage is also described. Finally, Alternate Protocol 2 describes how to induce noncanonical NLRP3 inflammasome activation by high‐dose LPS challenge in a sepsis model. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Priming and activation of inflammasomes in mouse macrophagesBasic Protocol 2 : Activation of human NLRP1 inflammasome by DPP8/9 inhibitor talabostatBasic Protocol 3 : Purification and detection of ASC pyroptosomeAlternate Protocol 1 : Detection of ASC speck by immunofluorescence stainingBasic Protocol 4 : Activation of canonical NLRP3 inflammasome in mice by intraperitoneal delivery of MSU crystalsAlternate Protocol 2 : Activation of noncanonical NLRP3 inflammasome in mice by intraperitoneal delivery of LPS -
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Abstract Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In
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Basic Protocol 1 : Long‐term auxin‐mediated depletion on platesSupport Protocol : Preparation of NGM and NGM‐auxin platesBasic Protocol 2 : Rapid auxin‐mediated depletion via soakingBasic Protocol 3 : Acute auxin‐mediated depletion in isolated embryosBasic Protocol 4 : Assessing auxin‐mediated depletion -
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Abstract In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus‐specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : LCMV infection routes in miceSupport Protocol 1 : Preparation of LCMV stocksASSAYS TO MEASURE LCMV TITERS Support Protocol 2 : Plaque assaySupport Protocol 3 : Immunofluorescence focus assay (IFA) to measure LCMV titerMEASUREMENT OF T CELL AND B CELL RESPONSES TO LCMV INFECTION Basic Protocol 2 : Triple tetramer staining for detection of LCMV‐specific CD8 T cellsBasic Protocol 3 : Intracellular cytokine staining (ICS) for detection of LCMV‐specific T cellsBasic Protocol 4 : Enumeration of direct ex vivo LCMV‐specific antibody‐secreting cells (ASC)Basic Protocol 5 : Limiting dilution assay (LDA) for detection of LCMV‐specific memory B cellsBasic Protocol 6 : ELISA for quantification of LCMV‐specific IgG antibodySupport Protocol 4 : Preparation of splenic lymphocytesSupport Protocol 5 : Making BHK21‐LCMV lysateBasic Protocol 7 : Challenge modelsTRANSGENIC MODELS Basic Protocol 8 : Transfer of P14 cells to interrogate the role of IFN‐I on CD8 T cell responsesBasic Protocol 9 : Comparing the expansion of naïve versus memory CD4 T cells following chronic viral challenge
