title: Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).
abstract: Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities.
authors.affiliation: Graduate Program in Marine Biology, Grice Marine Laboratory, College of Charleston, 205 Fort Johnson Rd., Charleston, SC 29412, United States; NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, 219 Fort Johnson Rd., Charleston, SC 29412, United States. Electronic address: meekme@g.cofc.edu.
authors.forename: Megan E
authors.initials: ME
authors.lastname: Meek
authors.affiliation: Graduate Program in Marine Biology, Grice Marine Laboratory, College of Charleston, 205 Fort Johnson Rd., Charleston, SC 29412, United States; NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, 219 Fort Johnson Rd., Charleston, SC 29412, United States.
authors.forename: Frances M
authors.initials: FM
authors.lastname: Van Dolah
mesh_terms: D004141:Dinoflagellida / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D010839:Phytoplankton / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity
chemical_list: D015800:Protozoan Proteins
keywords: Dinoflagellate; Flow cytometry; Immunofluorescence; In situ hybridization; Karenia brevis; Phytoplankton