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Abstract Orsay virus infection in the nematodeCaenorhabditis eleganspresents an opportunity to study host‐virus interactions in an easily culturable, whole‐animal host. Previously, a major limitation ofC. elegansas a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen,C. eleganshas emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability ofC. elegansenables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection ofC. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: SynchronizeC. elegansdevelopment by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT‐PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus‐infected intestinal tissue
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An Optimized Whole‐Mount Immunofluorescence Method for Shoot Apices
Abstract 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 (Zea mays) and Arabidopsis. We present the whole‐mount immunofluorescence procedure using maize ear primordia and Arabidopsis inflorescence apices as examples, followed by tips and suggestions for each step. In addition, we provide a supporting protocol to describe the use of an ImageJ plug‐in to analyze colocalization. This protocol has been optimized to observe proteins in 2‐5 mm maize ear primordia or in developing Arabidopsis inflorescence apices; however, it can be used as a reference to perform whole‐mount immunofluorescence in other plant tissues and species. © 2021 Wiley Periodicals LLC. Basic Protocol: Whole‐mount immunofluorescence for maize and Arabidopsis shoot apices Support Protocol: Measure colocalization by JACoP plugin in ImageJ
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- 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
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