The CARD-FISH procedure was performed with Alexa 488-labeled tyramides (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), as previously described (56 (link)), and analyzed manually using 10 to 20 microphotographs randomly taken by epifluorescence microscopy at ×1,000 magnification (AxioVision.M1; Carl Zeiss, Jena, Germany). Biovolume was estimated by multiplying cell abundance by average cell volume, which was calculated based on manual measurements of cell width and length and assuming the cell shape to be prolate spheroid, as described by Piwosz in 2019 (46 (link)). The relative abundance of an individual lineage was calculated as the proportion of cells hybridized with the specific probe to that of cells hybridized with the general eukaryotic probe. A full list of applied probes (n = 11) can be found in Table S2 .
Alexa 488 labeled tyramides
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 488-labeled tyramides are a type of fluorescent labeling reagent used in various biological and biochemical applications. They are designed to covalently attach to target molecules, enabling their detection and visualization through fluorescence microscopy or other fluorescence-based techniques. The core function of Alexa 488-labeled tyramides is to provide a fluorescent signal for the labeling and detection of target analytes.
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2 protocols using alexa 488 labeled tyramides
1
Quantifying Eukaryotic Diversity via CARD-FISH
2
Microbial Community Profiling by CARD-FISH
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The abundance of different microbial taxa in the water samples was determined with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH; Pernthaler et al., 2002) . The following probes were used to characterize the microbial community: EUB338 I-III (most bacteria; Daims et al., 1999) , ALF968 (most Alphaproteobacteria; Neef, 1997), BET42a (most Betaproteobacteria; Manz et al., 1992) , GAM42a (most Gammaproteobacteria; Manz et al., 1992) , CF319a (mainly groups of Cytophaga-Flavobacteria-Bacteroidete; Manz et al., 1996) .
Signal amplification was performed with Alexa488-labeled tyramides (Molecular Probes). CARD-FISH preparations were counterstained with DAPI at a final concentration of 1 μg mL -1 . DAPI-and CARD-FISH-stained cells were counted manually, achieving a minimum of 1000 cells per filter per evaluation.
Signal amplification was performed with Alexa488-labeled tyramides (Molecular Probes). CARD-FISH preparations were counterstained with DAPI at a final concentration of 1 μg mL -1 . DAPI-and CARD-FISH-stained cells were counted manually, achieving a minimum of 1000 cells per filter per evaluation.
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