title: Catalyzed reported deposition-fluorescence in situ hybridization protocol to evaluate phagotrophy in mixotrophic protists.
abstract: We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.
authors.affiliation: Unitat de Limnologia, Departament de Biogeoquimica Aquàtica, Centre d'Estudis Avançats de Blanes-CSIC, C/Accés a la Cala St. Francesc, 14, E-17300 Blanes (Girona), Spain. jmmedina@ceab.csic.es
authors.forename: Juan M
authors.initials: JM
authors.lastname: Medina-Sánchez
authors.forename: Marisol
authors.initials: M
authors.lastname: Felip
authors.forename: Emilio O
authors.initials: EO
authors.lastname: Casamayor
mesh_terms: D001105:Archaea / Q000235:genetics / Q000254:growth & development*; D001419:Bacteria / Q000235:genetics / Q000254:growth & development*; D044785:Cryptophyta / Q000502:physiology; D056890:Eukaryota / Q000235:genetics / Q000502:physiology*; D020387:Food Chain; D006735:Horseradish Peroxidase; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*
chemical_list: D015345:Oligonucleotide Probes; D006735:Horseradish Peroxidase