CARD-FISH

Catalyzed Reporter Deposition Fluorescence In-Situ Hybridization

According to Perntahler et al. (2002) with minor modifications


I. Preparation of filters

Boil 0.2% low-gelling point agarose in a microwave oven.

Put the filters with both sides into one drop of agarose and put the filters face-up onto the glass plate (using a separate drop for each filter avoids cell loss).

To remove the filters from the glass plate, pipet ethanol (96% to 80% [v/v]) onto the filters and carefully peel them off.

Let the filters air dry on paper tissue.

II. Inactivation of endogeneous peroxidases

Incubate filters in 0.01 M HCl at RT for 10 min.

Wash in MilliQ water.

Very important: test, if endogenous peroxidases have been bleached completely. If peroxidase activity is still present, the bleaching protocol needs to be optimized.


III. Permeabilization

The permeabilization procedure varies and needs to be optimized for each probe and sample!

After inactivation of endogenous peroxidases, try the treatments described below to permeabilize archaeal cell walls. A proper permeabilization of target cells is crucial for bright hybridization signals. Optimizing this step is highly recommended, e.g. by comparing each single treatment, varying single treatments (incubation time, concentrations) or the combinations of different treatments.

Incubate filter sections for 1 minute in 0.1 M HCl Rinse filters with excess water and dehydrate with Ethanol abs. Let filters air dry.

Incubate filter sections for 10 minutes in 1xPBS containing 1% Triton X-100 Rinse filters with excess water and dehydrate with Ethanol abs. Let filters air dry.

Incubate filter sections for 10 minutes in 1xPBS containing 0.5% SDS Rinse filters with excess water and dehydrate with Ethanol abs. Let filters air dry.

(continue with CARD-FISH methods on next page)

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