Updated 2012
M. Nikrad
CARD-FISH PROTOCOL
Catalyzed Reporter Deposition- Fluorescence In Situ Hybridization
(CARD-FISH)
Adapted from a protocol sent by Laura Alonso with notes by T. Straza
1. FIXATION (for planktonic bacteria and archaea).
• Add buffered particle-free paraformaldehyde (final conc 2%) to
sample and fix at 4ºC for 24 h.
• Filter the samples gently (5mm Hg) onto a white polycarbonate
filters (pore size 0.2 μm GTTP). Use cellulose nitrate support
filters beneath the polycarbonate filters to improve the
distribution of cells.
• Wash twice with 3-5 ml of MilliQ water (or PBS).
• Dry and store the filters at –20ºC. These samples can be stored for
months, probably years.
2. EMBEDDING CELLS INTO AGAROSE
• Prepare 0.1% (wt/vol) low gelling point agarose (Metaphor
Catalog# 50180 by Lonza) in milliQ water in a beaker (about
20ml’s total, can reuse once within the week).
• Melt the agarose before each use in a microwave oven or hot plate
till boiling.
• Let it cool down to 35-40ºC, use a thermometer.
• Dip both sides of filters into the agarose. Coat well.
CARD-FISH
• Put the filters with the cell side face up onto parafilm on a glass
plate. (Try to dip the filters quickly and make sure that there is
agarose on all of the surface.)
• Let the filters dry in the oven (35ºC) for 10-30 minutes.
• Pipet ethanol (96-80% [v/v]) onto the filters and carefully peel them
off.
• Let the filters air dry face up on Whatman paper or tissue.
3. PERMEABILIZATION WITH LYSOZYME
• Prepare 20 ml of a fresh lysozyme (Sigma L7651-10G) solution in
the following order (1-2ml of solution per filter piece):
2 ml EDTA 0.5 M
2 ml 1 M Tris HCl, pH8
16 ml MilliQ water
2 ml Lysozyme working solution
Note: To make Lysozyme working stock, just mix 10ml MilliQ with
1g lysozyme powder, mix and aliquot into 2 ml tubes and freeze in a
-20C. Thaw one tube at a time as necessary for use.
• Incubate the filters in 2 ml lysozyme solution for 60 min at 37ºC
(using a microtitre well plate). (Follow directly with
achromopeptidase permeabilization if examining archaea, 30 mins
at 37C.)
• Wash the filters in a small beaker filled with MilliQ water, then in a
beaker filled with 95% of absolute ethanol, by dipping 3 times in
the water and then 3 times in the ethanol.
• Let filters dry cell-side up on Whatman paper.
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CARD-FISH
Optional: After permeabilization filters can be stored at –20ºC for
several weeks. Make sure they are very dry before freezing.
4. HYBRIDIZATION
• Cut filters in sections depending on the number of probes necessary
(keeping large filter pieces until this point minimizes curling of the
filter after agarose embedding, use 1/12th of a whole filter per
probe).
• Mix hybridization buffer and HRP labeled oligonucleotide probe
(we use 3.3ul of 25ng/ul probe dilution + 500ul hybridization
buffer in 500ul Ependorf tubes)
• Place the filters into probe and hybridization buffer, one filter per
tube.
• Hybridize on a rotation shaker (10 rpm) overnight at 35ºC (12-18
hrs most probes).
5. WASHING
• After hybridization, place each filter section into 2 ml washing
buffer in 37C incubator for 40 minutes (use 12-well culture plate
for simplicity).
• Remove from wash buffer and use immediately in next step
• Do not let filter sections run dry, as this will reduce the activity of
the HRP. Put directly on a drop of Cy3 from the wash buffer (see
below)
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CARD-FISH
6. CATALYZED REPORTER DEPOSITION using TSA kit from Perkin
Elmer (NEL744)
• Prepare the amplification reagent: add Cy3 tyramide to
amplification reagent, 1:50. For example, 4 μl Cy3 tyramide plus
200 μl amplification reagent is enough for 6 filter pieces (30 ul per
filter). Amplification reagent is supplied with TSA kit.
• Place 30 μl Cy3 solution per filter piece on parafilm; place filter
piece cell-side down into the Cy3 solution.
• Incubate at room temperature in the dark for 3-10min.
• Remove excess liquid by gently dabbing filters onto blotting paper.
Wash sections in 1X PBS for 30 minutes at room temperature in
the dark.
• Wash sections in deionized water, then in 95% or absolute ethanol
by dipping. Let sections air dry on Whatman paper (keep in the
dark at this point and take to the dark-room for
microautoradiography if necessary). Otherwise,
• Place filter sections into 4 CITIFLUOR:1 VECTA containing
DAPI (1 μg/ml) and cover with cover slip for CARD FISH.
Sections could also be stored at –20ºC until further processing.
PREPARATION OF HYBRIDIZATION BUFFER
• Pipet into a 50 ml tube:
3.6 ml NaCl 5M
0.4 ml 1M Tris HCl
20 μl SDS of 1% stock
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CARD-FISH
14 ml of appropriate formamide solution (Concentration of
formamide depends on probe--see below)
2 ml blocking reagent at 1x
• Add 2 g of dextran sulfate. Heat at 40-60ºC and shake/rotate until
the dextran sulfate has dissolved completely.
Small portions of the buffer can then be stored at –20ºC for several
months.
Probe
% Formamide
Formamide (ml)
Water (ml)
NON338
20
4
10
Alphaproteobacteria, SAR11-441R,
45
9
5
NOR5
50
10
4
EUB338, CF319a, BET42a.
55
11
3
60
12
2
Ant4D3, Polaribacter740, SAR86
GAM42a, ROS537, Arctic96B16
ALT1416
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CARD-FISH
WASH BUFFER
Pipet in a 50mL tube:
0.5 ml EDTA
1 ml Tris HCl
5 M NaCl (Volume depends on probe, see below)
25 μl SDS (1% stock)
Add water to 50 ml.
% FORMAMIDE*
NaCl 5M (μL)
20%
1350
45%
160
50%
90
55%
30
60
0
*Used in hybridization buffer, but not in the wash buffer. The addition of NaCl
matches the formamide concentration in the hybridization buffer. Both NaCl
and formamide help to determine the stringency of the probe.
Probe concentrations
-1
The probe concentration depends on the probe; 3μl of probe (50 ng μl ) in
900μl of hybridization buffer is usually enough to detect CF, Roseobacter or
Gammaproteobacteria. Higher concentration of probes (9 μl of probe in 900μl
HB) is recommended for low-active cells or groups like Alphaproteobacteria ,
including SAR11. Adding more probe can increase the background.
Blocking Reagent
1. Buy "Blocking Reagent" from Boehringer Mannheim, although alternatives
(e.g. powdered milk) probably also work.
2. Mix up aliquots of 10X stock in 0.1M maleic acid and 0.25 M NaCl, pH 7.5
o
(see instructions from Boehringer) The 10X aliquots are stored at -20 C.
3. Dilute with MilliQ water to the 1X concentration used in the protocol.
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