II. Materials and methods
II.10 Fluorescence in situ hybridization (FISH)
II.10.6 CARD-FISH
1x PBS Volume
20x PBS 15 ml
Distilled water 285 ml
2x saline-sodium citrate buffer (SSC) Volume
NaCl 17.53 g
Sodium citrate 8.82 g
Distilled water 800 ml
Adjust pH to 7.0 with HCl, fill up to 1 l with water, and autoclave
1x Maleic acid buffer Volume
Maleic acid 5.8 g
NaCl 4.4 g
Distilled water 500 ml
Adjust pH to 7.5 with NaOH pellets
10% Blocking reagent Volume
Blocking reagent (Roche) 50 g
1x Maleic acid buffer 500 ml
Stir and heat up, until powder is completely dissolved. Aliquot, autoclave and store at -20°C (or at 4°C when in use).
Hybridization buffer - 50% formamide Volume
5 M NaCl 3.6 ml
1 M Tris-HCl pH 8.0 0.4 ml
20% SDS 20 µl
10% Blocking reagent 2 ml
Dextran sulphate 2 g
Formamide 10 ml
Nuclease-free water 3.98 ml
Dissolve at 40-60°C in water bath, aliquot, and store at -20°C
Amplification buffer Volume
5 M NaCl 16 ml
20x PBS 2 ml
10% Blocking reagent 0.4 ml
Dextran sulphate 4 g
Nuclease-free water 21.5 ml
Dissolve at 40-60°C in water bath and store at 4°C
Hybridization buffer - 10% formamide Volume
5 M NaCl 0.9 ml
1 M Tris-HCl pH 8.0 0.1 ml
20% SDS 5 µl
Dextran sulphate 0.5 g
Formamide 500 µl
Nuclease-free water 3.495 ml
For CARD-FISH of archaeal 16S rRNA. Dissolve at 40-60°C in water bath, aliquot, and store at -20°C Washing buffer for 16S rRNA CARD Volume
5 M NaCl 4.4 ml
0.5 M Na-EDTA 0.5 ml
1 M Tris-HCl pH 8.0 1 ml
20% SDS 25 µl
Distilled water 44.075 ml
equivalent to 10% formamide in hybridization buffer
1 mg ml-1 DAPI solution Volume 4',6-diamidino-2-phenylindole (Sigma) 2 mg
Nuclease free water 2 ml
II.10.6.1.1 Synthesis of tyramide conjugates
CARD is based on the deposition of a large number of labeled tyramine molecules by peroxidase activity. HRP reacts with hydrogen peroxide and the phenolic part of labeled tyramide to produce a quinine-like structure bearing a radical on the C2 group. This
greatly enhanced FISH sensitivity as compared to probes with a single fluorochrome.
Synthesis of tyramide conjugates was performed based on a protocol by Pernthaler et al.
(2004) and is described below.
Synthesis of tyramide labeled with 5- (and 6-) carboxyfluorescein:
a) Active dye stock:
100 mg 5- (and 6-) carboxyfluorescein succinimidyl ester (Invitrogen) + 10 ml dimethylformamide
Don’t expose to light!
b) Tyramine HCl stock:
33 µl triethylamine
+ 3.3 ml dimethylformamide + 33 mg tyramine-HCl (Sigma)
Mix 10 ml of active dye stock with 3.3 ml of tyramine HCl stock. Incubate for 6-12 h in the dark at room temperature. Add 86.7 ml 96% (v/v) ethanol to achieve 1 mg active dye ml-1. Aliquot (20 µl - 1 ml) and dry overnight in SpeedVac. Store dried tyramide conjugates at -20°C. Dissolve tyramides in dimethylformamide.
Synthesis of tyramide labeled with Alexa546:
a) Active dye stock:
1 mg succinimidyl ester AlexaFluor546 (Invitrogen) + 100 µl dimethylformamide
Don’t expose to light!
b) Tyramine HCl stock:
10 µl triethylamine + 1 ml dimethylformamide + 10 mg tyramine-HCl (Sigma)
Add 14.7 µl of tyramine HCl stock to active dye stock. Incubate for 6-12 h in the dark at room temperature. Add 875.3 ml 96% (v/v) ethanol to achieve 1 mg active dye ml-1. Aliquot (50 µl) and dry overnight in SpeedVac. Store dried tyramide conjugates at -20°C. Dissolve tyramides in 50 µl of dimethylformamide.
II.10.6.2 CARD-FISH mRNA USCα pmoA
CARD-FISH was performed with RNA polynucleotide probe A189f/Forest675r-T3 (see II.10.1) targeting the pmoA transcripts of USCα to detect cells expressing this gene in expression clones (induced and uninduced, see II.10.2), pure cultures, cells extracted from Marburg forest soil (see II.10.3), and in incubations of this soil on filters of soil substrate membrane systems (see II.11.1). The protocol is described below.
mRNA CARD-FISH of USCα pmoA:
1. Pretreatment
1. Add 5-10 µl of sample or a small filter piece to each well of a Teflon-coated glass slide (MAGV)
2. Dry slide for 5-10 min at 46°C
3. Boil up 0.1% low gelling point agarose (0.02 g in 20 ml distilled water), pour in Petri dish, let cool down to 35-40°C, and cover slide with agarose
4. Dry slide for 15-20 min at 46°C
5. Dehydrate slide in 50 ml 96% (v/v) ethanol for 1 min at room temperature 6. Air dry slide
7. Cover slide with 1 ml of 0.2 M HCl (see table II.10-6) and incubate for 10 min at room temperature
8. Wash slide in 50 ml 1x PBS for 1 min at room temperature
9. Incubate slide in 50 ml of 0.1% DEPC (50 µl DEPC + 50 ml 1x PBS, for carboxyethylation) for 12 min at room temperature
10. Wash slide in 50 ml 1x PBS for 1 min at room temperature 11. Wash slide in 50 ml distilled water for 1 min at room temperature 2. Permeabilization
1. Cover slide with 1 ml of lysozyme solution (5 mg ml-1 in 1x TE buffer) and incubate for 30 min at room temperature
2. Wash slide in 50 ml distilled water for 1 min at room temperature
3. Cover slide with 1 ml of proteinase K solution (1 µg ml-1 in 1x TE buffer) and incubate for 15 min at room temperature not for clones!
4. Wash slide 3 times in 50 ml distilled water for 1 min at room temperature 5. Dehydrate slide in 50 ml 96% (v/v) ethanol for 1 min at room temperature
6. Air dry slide 3. CARD-FISH
1. Cover slide with 100 µl of hybridization buffer (50% formamide, see table II.10-6) 2. Prepare wet chamber soaked with 2 ml of 50% formamide-1x SSC (1 ml
formamide + 1 ml 2x SSC)
3. Prehybridize slide in wet chamber for 1h at 58°C in hybridization oven
4. Mix 100 µl of hybridization buffer with 50 ng of RNA polynucleotide probe (~ 2-4 µl), denature mixture for 5 min at 80°C
5. Add probe mixture to wells (final probe concentration: 250 ng ml-1) 6. Prepare wet chamber soaked with 1 ml of hybridization buffer 7. Hybridize slide in wet chamber overnight at 58°C in hybridization oven
8. Wash slide in 50 ml of 50% formamide-1x SSC (25 ml formamide + 25 ml 2x SSC) for 1 h at 58°C
9. Wash slide in 50 ml of 0.2xSSC-0.01% (w/v) SDS (5 ml 2x SSC + 25 µl 20% SDS + 44.975 ml distilled water) for 30 min at 58°C
10. Incubate slide in 50 ml of 1x PBS-0.5% blocking reagent (2.5 ml 20x PBS + 2.5 ml 10% blocking reagent + 45 ml distilled water) for 30 min at room temperature 11. Mix 975 µl 1x PBS with 10 µl 10% blocking reagent and 15 µl of anti-DIG-HRP
antibody (= 0.75 U ml-1, Roche), add mixture on slide, and incubate for 1 h at 37°C
12. Wash slide 3 times in 50 ml 1x PBS for 10 min at room temperature
13. Mix 1 ml of amplification buffer (see table II.10-6) with 10 µl 100x H2O2 stock (1 ml 1x PBS + 5 µl 30% H2O2, Sigma Aldrich) and 5 µl of fluorescein labeled tyramide (see II.10.6.1.1)
14. Add mixture on slide and incubate for 5 min at room temperature in the dark 15. Wash slide in 50 ml 1x PBS for 3 min at room temperature
16. Wash slide 3 times in 50 ml distilled water for 1 min at room temperature 17. Dehydrate slide in 50 ml 50% (v/v) ethanol for 1 min at room temperature 18. Dehydrate slide in 50 ml 96% (v/v) ethanol for 1 min at room temperature 19. Air dry slide in the dark
4. DAPI staining
1. Add 20 µl of DAPI solution (see table II.10-6) per well and incubate for 10 min at room temperature
2. Wash slide in 50 ml distilled water for 1 min at room temperature
3. Dehydrate slide in 50 ml 96% (v/v) ethanol for 1 min at room temperature 4. Air dry slide in the dark
5. Continue with epifluorescence microscopy (see II.10.7)
II.10.6.3 CARD-FISH mRNA archaeal amoA
CARD-FISH was performed with RNA polynucleotide probe amo111f/amo643r-T3 (see II.10.1) targeting the amoA transcripts of ammonia-oxidizing archaea to detect cells expressing this gene in expression clones (induced and uninduced, see II.10.2) and in incubations of Rauischholzhausen agricultural soil incubated for stable isotope probing (see II.10.2). The protocol is described in II.10.6.2 with the following modifications:
2. Permeabilization
3. Cover slide with 1 ml of proteinase K solution (15 µg ml-1 in 1x TE buffer) and incubate for 10 min at room temperature for soil samples
3. CARD-FISH hybridization and washing temperature was 60.5° instead of 58°C!
II.10.6.4 CARD-FISH mRNA hydB
CARD-FISH was performed with RNA polynucleotide probe NiFe-1129f/NiFe-1640r-T3 (see II.10.1) targeting the hydB transcripts of Streptomyces sp. PCB7 to detect cells expressing this gene in expression clones (induced and uninduced, see II.10.2) and pure cultures. The protocol is described in II.10.6.2 with the following modifications:
2. Permeabilization for Streptomyces sp. PCB7
1. Cover slide with 1 ml of lysozyme solution (10 mg ml-1 in 1x TE buffer) and incubate for 1 h at 37°C
3. Cover slide with 1 ml of achromopeptidase solution (60 U ml-1 in 0.01 M NaCl and 0.01 M Tris-HCl; Sigma) and incubate for 30 min at 37°C
II.10.6.5 CARD-FISH 16S rRNA archaea
CARD-FISH of archaeal 16S rRNA was attached to CARD-FISH of archaeal amoA transcripts. HRP-labeled probe Arch915 (Biomers, Ulm, Germany) was used for detection of Archaea in soil samples with Alexa546-labeled tyramide. The protocol was inserted after step 3.17 of the mRNA CARD-FISH protocol (see II.10.6.3) and is described below.
CARD-FISH of archaeal 16S rRNA attached to mRNA CARD-FISH:
1. Incubate slide in 0.01 M HCl for 10 min at room temperature (to inactivate anti-DIG-HRP antibody)
3. Wash slide in 50 ml distilled water for 1 min at room temperature
4. Dehydrate slide in 50 ml 50% (v/v) ethanol for 1 min at room temperature 5. Dehydrate slide in 50 ml 96% (v/v) ethanol for 1 min at room temperature 6. Air dry slide in the dark
7. Mix 200 µl of hybridization buffer (10% formamide, see table II.10-6) with 2 µl of probe working solution of HRP-labeled probe Arch915 ( 50 ng µl-1) and add 20 ml of solution to each well
8. Prepare wet chamber with 1 ml of hybridization buffer (10% formamide) 9. Hybridize slide in wet chamber for 2.5 h at 46°C in hybridization oven
10. Wash slide in prewarmed washing buffer (see table II.10-6) for 15 min at 48°C 11. Dip slide shortly in cold water (4°C)
12. Wash slide in 50 ml 1x PBS for 15 min at room temperature
13. Mix 1 ml of amplification buffer (see table II.10-6) with 5 µl of Alexa546 tyramide (1 mg ml-1, see II.10.6.1.1) and add mixture on slide.
14. Incubate slide for 10 min at room temperature
15. Wash slide in 50 ml 1x PBS for 10 min at room temperature 16. Wash slide in 50 ml distilled water for 1 min at room temperature
17. Dehydrate slide in 50 ml 96% (v/v) ethanol for 1 min at room temperature 18. Air dry slide in the dark
19. Continue with DAPI-staining (mRNA CARD-FISH protocol, step 4.1)