PhageFISH for DIG-labelled bacterial probes

Place liquid sample in a 30-50µL droplet on poly-L-lysine coated slide.

Dry in warm incubator for approx. 0h 30m 0s or until the droplet has dried out.

OPTIONAL: if sample is very dilute add several droplets and repeat drying procedure.

Add 1% paraformaldehyde to cover the sample area.

Incubate at Room temperature for 1h 0m 0s.

Aspirate the paraformaldehyde off.

Rinse samples in PBS for 0h 1m 0s.

Mix a small faecal sample with 10-20µL PBS (1X) and vortex thoroughly.

Allow suspension to settle for 0h 5m 0s.

Take 10µL of the supernatant and place on coated glass slide.

Smear the droplet over the slide using a cover slip.

Allow the sample to dry – this should not take more than 0h 10m 0s.

Overlay the slides with 1% paraformaldehyde. Ensure the whole sample area is covered (approx. 1mL).

Incubate for 1h 0m 0s at Room temperature or at 4°C.

Aspirate off excess paraformaldehyde.

Wash in PBS for 0h 1m 0s.

Add lysozyme to permeabilisation buffer.

Overlay samples with permeabilisation buffer.

Incubate On ice for 1h 0m 0s.

Wash samples in PBS for 0h 5m 0s.

Wash samples in sterile water for 0h 1m 0s.

Incubate samples in 0.01Molarity (M) HCl for 0h 10m 0s.

Wash samples in PBS for 0h 5m 0s.

Wash samples in sterile water for 0h 1m 0s.

Wash samples in 96% ethanol for 0h 1m 0s.

Allow slides to dry on blotting paper or filter paper.

Place filters in a petri dish and spot up to 100µL hybridisation buffer to cover the filters.

Transfer to a humidity chamber with hybridisation buffer soaked paper towels.

Incubate for 1h 0m 0s at hybridisation temperature______.

Mix 1mL gene hybridisation buffer with 1µL of each probe. Vortex to mix.

Place one droplet of 30-100µL probe mix on a petri dish for each filter.

Place the filters face down in the probe mix droplets.

Place the dish back in the humidity chamber and incubate for 1h 0m 0s at 85°C.

Immediately place the humidity chamber at hybridisation temperature 1h 0m 0s.

Wash filters.

Wash filters in gene washing buffer I 0h 1m 0s. (1/3)

Wash filters in gene washing buffer I 0h 1m 0s. (2/3)

Wash filters in gene washing buffer I 0h 1m 0s. (3/3)

Wash filters in gene washing buffer I 0h 30m 0s at 42°C.

Wash filters.

Wash filters in gene washing buffer II for 0h 1m 0s. (1/3)

Wash filters in gene washing buffer II for 0h 1m 0s. (2/3)

Wash filters in gene washing buffer II for 0h 1m 0s. (3/3)

Wash filters in gene washing buffer II for 1h 30m 0s at 42°C.

Wash filters in PBS for 0h 1m 0s.

Place filters in a petri dish and add antibody blocking solution to cover the filters. Incubate for 0h 30m 0s.

Move filters to antibody binding solution and incubate for 1h 30m 0s.

Wash filters.

Wash filters in antibody washing solution for 0h 1m 0s .

Wash filters in antibody washing solution for 0h 10m 0s. (1/3)

Wash filters in antibody washing solution for 0h 10m 0s. (2/3)

Wash filters in antibody washing solution for 0h 10m 0s. (3/3)

Mix 1mL amplification buffer with 10µL H₂O₂ and 2µL Alexa tyramides (488). Vortex to mix.

Place filters in a petri dish and cover with probe mix by spotting droplets of 30-100µL.

Wash filters.

Wash filters in PBS for 0h 1m 0s

Wash filters in PBS for 0h 5m 0s.

Wash filters in PBS for 0h 10m 0s at 46°C. (1/2)

Wash filters in PBS for 0h 10m 0s at 46°C. (2/2)

Wash filters in sterile water for 0h 1m 0s.

Wash filters in 96% ethanol for 0h 1m 0s.

Add 10.8mL sterile water, 1.2mL Tris-HCl (1M, pH 8), 15µL RNase I, and 30µL RNase A to a 15ml falcon tube.

Place filters in the RNase solution and incubate for 1h 0m 0s at 37°C.

Wash filters in PBS for 0h 5m 0s.

Repeat wash.

Wash filters in sterile water for 0h 1m 0s.

Cover samples with hybridisation buffer.

Transfer to a humidity chamber with formamide soaked paper towels at the corresponding concentration.

Incubate for 1h 0m 0s at hybridisation temperature (approx. 46°C).

Mix 1mL gene hybridisation buffer with 1µL of each probe. Vortex to mix.

Cover the samples with the hybridisation buffer-probe mix.

Place the dish back in the humidity chamber and incubate for 1h 0m 0s at 85°C.

Immediately place the humidity chamber at hybridisation temperature 1h 0m 0s.

Wash filters.

Wash filters in gene washing buffer I for 0h 1m 0s. (1/3)

Wash filters in gene washing buffer I for 0h 1m 0s. (2/3)

Wash filters in gene washing buffer I for 0h 1m 0s. (3/3)

Wash filters in gene washing buffer I for 0h 30m 0s at 42°C.

Wash filters.

Wash filters in gene washing buffer II for 0h 1m 0s. (1/3)

Wash filters in gene washing buffer II for 0h 1m 0s. (2/3)

Wash filters in gene washing buffer II for 0h 1m 0s.(3/3)

Wash filters in gene washing buffer II for 1h 30m 0s at 42°C.

Wash filters in PBS for 0h 1m 0s.

Place filters in a petri dish and add antibody-blocking solution to cover the filters. Incubate for 0h 30m 0s.

Move filters to antibody binding solution and incubate for 1h 30m 0s.

Wash filters.

Wash filters in antibody washing solution for 0h 1m 0s.

Wash filters in antibody washing solution for 0h 10m 0s. (1/3)

Wash filters in antibody washing solution for 0h 10m 0s. (2/3)

Wash filters in antibody washing solution for 0h 10m 0s. (3/3)

Mix 1mL amplification buffer with 10µL H₂O₂ and 2µL Alexa tyramides (594). Vortex to mix.

Place filters in a petri dish and cover with probe mix by spotting droplets of 30-100µL. Incubate at 37°C for 0h 45m 0s.

Wash filters.

Wash filters in PBS for 0h 1m 0s.

Wash filters in PBS for 0h 5m 0s.

Wash filters in PBS for 0h 10m 0s at 46°C.

Wash filters in PBS for 0h 10m 0s at 46°C.

Wash filters in sterile water for 0h 1m 0s.

Wash filters in 96% ethanol for 0h 1m 0s.

Mix 1mL SlowFade Gold with 1µL 5mg/mL DAPI dye.

Apply 5-10µL mix in droplets to each slide.

Apply coverglass and carefully press down to seal sample with minimal air bubbles.

Seal with clear nail polish on all edges of the sample.

Allow to cure completely.

Store at -20°C.