Table 1 Different steps of the followed CARD-FISH protocol.

From: Endosymbiotic calcifying bacteria across sponge species and oceans

Step

Description

1. Sample fixation

1. Incubate sponge samples within 4% paraformaldehyde solution for 4 h

2. Incubate samples in ethanol 70% during 18–24 h at 4 °C

3. Keep samples in ethanol 70% at −20 °C

2. Sample dehydration

Sequential incubation of sponge samples in ethanol 96% and 100%, ethanol:toluene (1:1) 30 min each, and absolute toluene 15 min

3. Embedding in paraffin and tissue sectioning

1. Include samples in paraffin at 50 °C for 24–48 h

2. Cut thick (6 μm) sections with an Autocut 2030 (Reichert-Jung) microtome and dry for 3 h at 40 °C

4. Deparaffinization

1. Incubate sections within Xylene for 10 min

2. Rehydratation by sequential incubation in ethanol 100%, 96%, 70%, 10 min each

3. Three baths in MiliQ water 5 min each, air dry

5. Membrane permeabilization

1. Incubate sections in 10 mg/ml Lysozime solution (Sigma USA), 0.05 M EDTA, 0.1 M Tris-HCl for 1 h at 37 °C

2. Wash with MiliQ water for 2 min, air dry

6. Endogenous peroxidases inactivation

1. Incubate sections in 0.1 M HCl solution for 30–60 sec

2. Wash with 1X PBS for 2 min

3. Incubate in 3% H2O2 solution for 10 min

4. Wash with MiliQ water and ethanol 96% 2 min each, air dry

7. Hybridization

1. Cover sections with hybridization buffer (1) solution together with the probe, helpers and competitors in a 3:100 volumetric ratio. Sequences (5′–3′) are:

CAL32L: CCCCTCTATCTGCGGCGG

Competitor I: CCCCTCTTTCTCCGGCGG

Competitor II: CCCCTCATTCTGCGGCGG

Helper 3′: YACAAGCTAATCGGACGCGGG

Helper 5′: YACAAGCTAATCGGACGCGGG

2. Incubate at 46 °C for 5 h with a solution of 45% formamide in humid chambers.

3. Wash the sections with pre-warmed washing buffer (2) at 48 °C for 10 min

8. CARD

1. Wash sections in 1X PBS (pH = 8) for 15 min

2. Cover sections with primary CARD substrate mix (3)

3. Incubate at 46 °C for 20 min

4. Wash twice in 1X PBS for 6 min at 46 °C and RT

5. Wash twice in MiliQ water for 2 min each and air dry.

  1. (1) The hybridization buffer was made by mixing 5 M NaCl, 1 M Tris-HCl (pH7.5), 20% sodium dodecyl sulfate (SDS), 10% (w/v) dextran sulfate, 10% Blocking Reagent and 45% formamide (Sigma). (2) Fresh whashing buffer was prepared by mixing 0.5 M EDTA, 1 M Tris-HCl, NaCl and 20% SDS in steryle miliQ water and warmed at 48 °C previously to the wash step. (3) The primary CARD substrate was made by mixing amplification buffer (10% (w/v) dextran sulfate, 2 M NaCl, 0.1% (w/v) blocking reagent, in 1X PBS (pH = 8)) with a freshly prepared H2O2 solution (0.15% in 1X PBS) at a ratio of 100:1. The needed volume of that primary CARD mix solution was mixed with 1 mg dye ml-1 tiramide-Alexa488 solution (Molecular Probes, Inc., Eugene, OR, USA) at a ratio of 500:5.
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