PubMed · 14626435
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Isaac D Montoya. 2003. Effecting translational research practices.. https://pubmed.ncbi.nlm.nih.gov/14626435/
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The low-fading immunofluorescence with propidium iodide contrast described here is recommended for light and confocal viral antigen identification and other cell biology studies because: (1) it is a simple, rapid, sensitive, and reproducible technique; (2) phase-contrast microscopy is unnecessary; (3) contrast is optimal without blurring the fluorescent labeling; (4) autofluorescence is minimal, even in fixed cells; (5) background staining is minimal; (6) fading is invisible for at least 5-min exposures, even in preparations with weak antigen presentation; (7) fluorescence is stable after storage in the dark at -20 degrees C; (8) fluorochromes are small-sized markers without steric hindrance; and (9) there is no need for silver enhancement or substrate solutions, which increase the risk of diffusion and other artifacts.
The use of tyramide-coupled immunofluorescence at the single cell level provides expedient, clean, and sensitive signals for detection of DNA, RNA, or proteins. The principle is based on the ability of horseradish peroxidase (HRP) to cleave tyramides into a free radical species with a very short diffusion radius. The free radicals are then covalently bound to electron-rich moieties such as tyrosine in proteins proximal to the targets. Here we present protocols for tyramide fluorescent in situ hybridization (T-FISH), which detects unique DNA species using DNA probes as short as approx 300-500 bp, or unique RNA species with probes as small as an oligonucleotide. We also present a protocol for tyramide immunofluorescence (T-IF) to detect protein antigens. By combining these protocols with several tyramide-coupled fluorophores, multiple targets can be detected simultaneously in situ, which is ideal for in-depth analyses at the molecular and cellular levels. Finally, we describe the detection of nascent viral RNA transcripts simultaneously with integrated viral genomes or chromosomal domains in single cells or tissue sections.
In 1988, the World Health Assembly resolved to eradicate poliomyelitis globally by 2000. Progress toward achieving this goal has been reported from countries where polio is endemic, and three World Health Organization (WHO) regions (Americas, Europe, and Western Pacific) appear to be free of indigenous wild poliovirus (WPV) transmission. One key strategy for eradicating polio is establishing sensitive polio surveillance systems by investigating acute flaccid paralysis (AFP) cases. To ensure that specimens from persons with AFP undergo appropriate processing for viral isolation, WHO established a global polio laboratory network in 1988. This report updates previous publications, summarizes the laboratory network's performance, and describes the location and characterization of WPV and vaccine-derived poliovirus (VDPV) during January 2003-June 2004.