Visualization of gene products in
The localization of a protein provides important information about its biological functions. The visualization of proteins by immunofluorescence has become an essential approach in cell biology. Here, we describe an easy‐to‐follow immunofluorescence protocol to localize proteins in whole‐mount tissues of maize (
- NSF-PAR ID:
- 10220824
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 1
- Issue:
- 4
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single‐molecule fluorescencein situ hybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in theC. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH inC. elegans embryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in‐tube approach for antibody staining that negates freeze‐cracking, a validated method to perform the cost‐reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single‐molecule RNA and protein detection. Moreover, simultaneous, high‐resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC.Basic Protocol 1 : Sequential immunofluorescence and single‐molecule fluorescencein situ hybridizationAlternate Protocol : Abbreviated protocol for simultaneous immunofluorescence and single‐molecule fluorescencein situ hybridizationBasic Protocol 2 : Simplified immunofluorescence inC. elegans embryosBasic Protocol 3 : Single‐molecule fluorescencein situ hybridization or single‐molecule inexpensive fluorescencein situ hybridization -
Abstract Cross‐presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC‐I molecules to cross‐prime CD8+T cells has increased significantly. Dendritic cells (DCs) are exceptional cross‐presenters, thus making them a great tool to study cross‐presentation but the relative rarity of DCs in circulation and in tissues makes it challenging to isolate sufficient numbers of cells to study this process in vitro. In this paper, we describe in detail two methods to culture DCs from bone‐marrow progenitors and a method to expand the numbers of DCs present in vivo as a source of endogenous bona‐fide cross‐presenting DCs. We also describe methods to assess cross‐presentation by DCs using the activation of primary CD8+T cells as a readout. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Isolation of bone marrow progenitor cellsBasic Protocol 2 : In vitro differentiation of dendritic cells with GM‐CSFSupport Protocol 1 : Preparation of conditioned medium from GM‐CSF producing J558L cellsBasic Protocol 3 : In vitro differentiation of dendritic cells with Flt3LSupport Protocol 2 : Preparation of Flt3L containing medium from B16‐Flt3L cellsBasic Protocol 4 : Expansion of cDC1s in vivo for use in ex vivo experimentsBasic Protocol 5 : Characterizing resting and activated dendritic cellsBasic Protocol 6 : Dendritic cell stimulation, antigenic cargo, and fixationSupport Protocol 3 : Preparation of model antigen coated microbeadsSupport Protocol 4 : Preparation of apoptotic cellsSupport Protocol 5 : Preparation of recombinant bacteriaBasic Protocol 7 : Immunocytochemistry immunofluorescence (ICC/IF)Support Protocol 6 : Preparation of Alcian blue‐coated coverslipsBasic Protocol 8 : CD8+T cell activation to assess cross‐presentationSupport Protocol 7 : Isolation and labeling of CD8+T cells with CFSE -
Abstract Background In the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported.
Results We adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol.
Conclusions The whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.
-
Abstract Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In
Caenorhabditis elegans , a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation.C. elegans is also a powerful genetic organism, amenable to large‐scale genetic screens and CRISPR‐mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin‐inducible degradation. In order to apply this approach inC. elegans , a 44–amino acid degron tag is added to the protein of interest, and theArabidopsis ubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron‐tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin‐mediated depletion of target proteins inC. elegans , highlighting the versatility and power of this method. © 2021 Wiley Periodicals LLC.This article was corrected on 19 July 2022. See the end of the full text for details.
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 -
Abstract Mass spectrometry‐based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly
Pseudomonas aeruginosa , and the formation of biofilms. The interplay betweenP. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil‐associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced byP. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC.Basic Protocol 1 : Murine model of ocular infectionBasic Protocol 2 : Murine model sample collectionBasic Protocol 3 : Protein extraction from eye washBasic Protocol 4 : Protein extraction from corneal tissueBasic Protocol 5 : Mass spectrometry‐based proteomics and bioinformatics from eye wash and corneal tissue samples