TABLE 2.

Proposed CARD-FISH protocol for evaluation of mixotrophic protists phagotrophy on Archaea (HRP-ARCH915 probe) and Bacteria (HRP-EUB338 probe)

Stage	Step	Descriptiona
Sample preparation	1	Take aliquot (150 ml) from sample and fix it with Lugol's solution. Let it settle onto a Uthermöl chamber and visualize it by using an inverted microscope.
	2	Fix sample with 0.5% (vol/vol) alkaline Lugol's solution, followed by 2% buffered formaldehyde, and within a 2- to 3-min interval add several drops of 3% sodium thiosulfate to decolorate Lugol (fixation time, 1 h maximum).
	3	Filter using a vacuum pump (<100 mm Hg) separate aliquots (volume for a suitable number of cells homogeneously distributed throughout filter depends on their initial abundance) onto 1- to 2-μm and 0.2-μm-pore-size polycarbonate filters, respectively.
	4	Wash twice with 10 ml of sterile ddH2O.
	5	Let air dry.b
Embedding	6	Dip filters in 0.1 to 0.2% (wt/vol) low-gelling-point agarose at 35 to 40 °C.
	7	Place filters face down onto parafilm or glass plate, let them dry at 35 to 40°C (10 to 20 min).
	8	Remove filters by soaking them in 80 to 96% ethanol (RT).
	9	Let air dry.b
Permeabilization	10	Incubate filters in fresh lysozyme solution (10 mg ml−1 FC, dissolved in 0.05 M EDTA [pH 8.0], 0.1 M Tris-HCl [pH 7.4]) for 60 min at 37°C.
	11	Wash with ddH2O (1 min, RT).
	12	Incubate filters in fresh achromopeptidase solution (60 U ml−1 FC, dissolved in 0.01 M NaCl, 0.01 M Tris-HCl [pH 8.0]) for 30 min at 37°C.
	13	Wash with ddH2O.
	14	Incubate filters in 0.01 M HCl for 10 min at RT to inactivate endogenous peroxidase.
	15	Wash thoroughly with ddH2O.
	16	Dehydrate in ethanol series (50, 80, 100%, 3 min each at RT).
	17	Let air dry.b
Hybridization	18	Place filters in an adapted Ecostep syringe.
	19	Mix (100:1 [vol/vol]) hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 7.4], 10% [wt/vol] dextran sulfate, 0.02% [wt/vol] sodium dodecyl sulfate, 55% [vol/vol] formamide, 1% blocking reagent prepared in maleic acid buffer [100 mM maleic acid, 150 mM NaCl; pH 7.5]) and probe solution (50 ng μl−1 sterile ddH2O) at a suitable quantity to dip filters after adjusting the piston.
	20	Incubate filters at 35°C for 4 h on a rotation shaker.
	21	Wash filters in prewarmed washing buffer (20 mM NaCl, 5 mM EDTA [pH 8.0], 20 mM Tris-HCl [pH 7.4], 0.02% [wt/vol] sodium dodecyl sulfate) for 5 to 10 min at 37°C. Do not air dry filters after washing.
CARD	22	Incubate in 1× PBS (0.2-μm filtered) amended with 0.05% of Triton X-100 (15 min at RT).
	23	Remove excess liquid with blotting paper, but do not let filters dry.
	24	Incubate in substrate mix (1 part Alexa488-labeled tyramide [1 mg ml−1 FC dissolved in dimethylformamide containing 20 mg ml−1p-iodophenylboronic acid] and 100 parts amplification buffer [10% {wt/vol} dextran sulfate, 2 M NaCl, 0.1% {wt/vol} blocking reagent, and freshly prepared 0.0015% H2O2 in 1× PBS pH 7.6]) for 15 min at 37°C in the dark.
	25	Dab filters in blotting paper.
	26	Wash in 1× PBS amended with 0.05% Triton X-100 (15 min, RT).
	27	Wash in ddH2O (1 min, RT).
	28	Wash in 96% ethanol (1 min, RT).
	29	Let air dry.b
Mounting	30	Counterstain filters by soaking them with DAPI solution (1 μg ml−1 FC) and mount filters on slides using Citifluor AF1.

^addH₂O, double-distilled water; RT, room temperature; FC, final concentration.

^bPreparations may be stored at −20°C for several weeks without loss in signal.