Table 2.

Basic protocol for CARD-FISH for rRNA

Cell fixation 1 Fix samples with paraformaldehyde or ethanol, and store in ethanol/PBS soltion at −20°C. Embedding  on glass slide 2-1 Mix the samples with PBS, SDS (final conc., 0.001%), and low-melting point agarose (final conc., 0.1%), apply appropriate amount to each well, and dry at 35–60°C. 2-2 Dehydrate and desalt through ethanol series (50% for 5 min, 80% for 1 min, and 96% for 1 min), and air dry.  on membrane filter 2-1 Filter the samples through membrane filters. 2-2 Dip the filters in 0.1–0.2% low-melting-point agarose, place them upside-down on Parafilm, and dry at 35–60°C. 2-3 Place ethanol on the filters to detach from Parafilm, and air dry. 2-4 Cut the filters, if necessary Permeabilization 3-1 Lysozyme, lysozyme + achromopeptidase, proteinase K, pseudomurein endopeptidase, HCl, or microwave treatments, based upon your interests. 3-2 Wash in TNT (100 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20) or PBSX (0.05% Triton X-100 in PBS) for 5–15 min at room temperature (RT). 3-3 Wash in ultra-pure water for 1 min, dehydrate in ethanol for 1 min, and air dry. Endogenous peroxidase inactivation 4-1 0.15% H2O2 in methanol, diethylpyrocarbonate, and/or HCl treatments depending on your samples. 4-2 Wash in TNT or PBSX for 5–15 min at RT, if necessary. 4-3 Wash in ultra-pure water for 1 min, dehydrate in ethanol for 1 min, and air dry. In situ hybridization and washing 5-1 Prepare hybridization buffer: e.g., 0.9 M NaCl, 20 mM Tris-HCl [pH 7.2], 10% dextran sulphate, 1% blocking reagent, 0.01% SDS, and formamide (concentration depends on the probe) 5-2 Mix probe with hybridization buffer at a final concentration of 0.1 μM. 5-3 Incubate at 35–46°C for more than 2 hours. 5-4 Prepare washing buffer: e.g., 0.9 M NaCl, 20 mM Tris-HCl [pH 7.2], 5 mM EDTA, 0.01% SDS, and formamide. 5-5 Dip the glass slides or the filters in washing buffer and incubate for 10–20 min at 2°C above the hybridization temperature. Catalyzed reporter deposition (tyramide signal amplification) 6-1 Dip the glass slides or the filters in TNT or PBSX for 15 min at RT. Do not air dry. 6-2 Prepare tyramide working solution: e.g., one volume of tyramide and 50–100 volumes of amplification buffer (0.0015% H2O2, 0.1% blocking reagent, 10–20% dextran sulphate in PBS) or if using a kit from PerkinElmer, one volume of tyramide, 37.5 volumes of amplification diluent, 12.5 volumes of 40% dextran sulphate, and 0.5 volumes of 10% blocking reagent. 2 M NaCl and p-Iodophenylboronic acid (20:1 to tyramide concentration) can be added to further enhance signals. 6-3 Incubate with tyramide working solution for >15 min at 37°C. 6-4 Wash in TNT or PBSX for 15 min at RT or elevated temperature of 45–55°C. 6-5 Wash in ultra-pure water for 1 min, dehydrate in ethanol for 1 min, and air dry.