Whole cell hybridization The different steps

Filtration and permeabilization Filter the samples on Anodisc filters (0.22  m) Deshydrate the cells with immerse in alcohol Cut the filters in 12 pieces Put the pieces at -80°C

Hybridization Prepare the humid chamber and put it at 35°C to equalize the temperature Put 9  L of hybridization buffer and 1  L of specific probes on the slide Transfer the slides into the humid chamber Incubate 2h to 3h at 35C

Wash Immerse the filters in plastic wells in 5 mL Wash buffer Cover with plastic lid and incubate 2 times 30 min at 37°C

Equilibration Immerse the filters in plastic wells in 5 mL TNT buffer Cover with plastic lid and incubate 15 min at room temperature, in dark

Enzymatic reaction (TSA) Put 10  L of the fluorochrome tyramide on the filters Incubate at room temperature in dark for 30 min

Wash Immerse the filters in plastic wells in 5 mL TNT buffer Cover with plastic lid and incubate 2 times 20 min at 55°C

DAPI coloration Put 10  L of DAPI (5  g/mL) on the slide Cover with filter Incubate for 15 min in dark at room temperature Rinse the filters in water during 10 min then allow the filter to dry on slide

Montage des lames Cover filters with 20  L of antifading citifluor agent Cover with cover slip Fix each slides of the cover slip with varnish

Stockage The slides are kept at 4°C in dark and can be observed after at least several days

Results Hybridization of Chlamydomonas concordia by the probe CHLO 01