TABLE 2.

Summary of steps used for FISH and BrdU detection in marine bacteria

StepProcedure
EmbeddingPrepare subsamples on membrane filters
Dip filters in 0.2% low-gelling-point agarose, place filters face up on glass slides and let air dry at 46°C
Dehydrate in 96% ethanol (1 min, RT), air drya
Permeabilization and epitope unmaskingIncubate in epitope retrieval buffer (60°C, 15 min), wash in MilliQ H2O
Wash in HCl (1 M) (60°C, 5 s), wash in MilliQ H2O
Incubate in permeabilization buffer (60°C, 5 min), wash in MilliQ H2O, wash in 96% ethanol, let air dry
Repeat embedding step
Incubate with lysozyme (10 mg/ml in 0.1 M Tris-HCl-0.05 M EDTA) (37°C, 60 min)
Wash in MilliQ H2O, wash in 96% ethanol, air drya
HybridizationPlace filter sections in reaction vial (1.5 ml, 10 to 20 sections per vial) and cover filter sections with 1,000 μl of hybridization buffer (0.5 ng of probe/μl), incubate at 35°C for 2 h
Wash filters in prewarmed washing buffer (5 min, 37°C)
Tyramide signal amplificationIncubate in PBST (50 ml, RT, 15 min), dab filter on blotting paper, but do not let run dry
Incubate filter sections in freshly prepared substrate mixture (1 part of Alexa488-tyramide, 100 parts of amplification buffer) (37°C, 10 min, in the dark)
Wash in PBST (RT, 5 min, in the dark), wash in MilliQ H2O (RT, 1 min), wash in ethanol (96%, RT, 1 min), air drya
Antibody reactionBleach peroxidases with 0.01 M HCl (RT, 10 min), wash three times in MilliQ H2O
Place filter sections on Parafilm and cover with 500 μl of anti-BrdU reaction mixture (1.5 U of anti-BrdU-HRP per ml, 1 × PBS, 1% blocking reagent, 20 U of HAEIII per ml), seal with Parafilm and incubate at 37°C for 2.5 to 3 h
Wash filter sections in PBST (10 min, RT, in the dark)
Tyramide signal amplificationDab filter sections on blotting paper but do not let run dry
Incubate in freshly prepared substrate mixture (1 part of Alexa536-tyramide, 200 parts of amplification buffer) (37°C, 10 min, in the dark)
Dab filter sections on blotting paper, wash in PBST (5 min, RT), wash in MilliQ H2O (RT, 1 min), wash in ethanol (96%, RT, 1 min), air drya
Embed samples and visualize
  • a Preparations may be stored at −20°C for several days to weeks without apparent loss of signal.