Whole cell hybridization

1. Whole cell hybridization The different steps

2. 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

3. 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

4. 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

5. 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

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

7. 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

8. 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

9. 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

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

11. Results • Hybridization of Chlamydomonas concordia by the probe CHLO 01