PhageFISH detailed protocol

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

Allow suspension to settle for 0h 5m 0s to avoid large debris.

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

Smear the droplet thinly over the slide using a cover slip.

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

Work in fume hood. Overlay the slides with 1% paraformaldehyde (PFA). Ensure the whole sample area is covered (approx. 1mL).

Incubate for 1h 0m 0s at Room temperature in the fume hood.

Aspirate off excess PFA.

Wash in PBS for 0h 1m 0s .

Add lysozyme to permeabilisation buffer.

Overlay samples with permeabilisation buffer.

Incubate On ice for 1h 0m 0s.

Discard permeabilisation buffer.

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.

Work in fume hood. Place a paper towel in the bottom of the hybridisation chamber and soak in formamide/milliQ solution corresponding to the hybridisation buffer concentration.

Overlay samples with hybridisation buffer-probe mix at 0.5ng/µl of each probe and close humidity chamber.

Incubate at 46°C for 3h 0m 0s.

Prepare the washing buffer – heat to 48°C.

Work in fume hood. Overlay the samples with washing buffer and incubate for 0h 15m 0s at 48°C (in humidity chamber to avoid formamide fumes).

Wash samples in sterile water.

Allow samples to dry.

Work in fume hood. Place a paper towel in the bottom of the hybridisation chamber and soak in formamide/milliQ solution corresponding to the hybridisation buffer concentration.

Overlay samples with hybridisation buffer (no probes!) and close humidity chamber (500µL per slide).

Incubate for 1h 0m 0s at 46°C.

Cover the samples with hybridisation buffer-probe mix at 10pg/µl of each probe (500µl per slide).

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 slides.

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

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

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

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

Wash slides.

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

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

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

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

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

Cover slides with antibody-blocking solution. Incubate for 0h 30m 0s.

Discard antibody-blocking solution and cover with antibody binding solution. Incubate for 1h 30m 0s.

Wash slides.

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

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

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

Wash slides 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.

Cover slides with CARD buffer-tyramide mix (approx. 500µL per slide). Incubate at 37°C for 0h 45m 0s.

Wash slides.

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

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

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

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

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

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

Mix 1mL SlowFade Gold antifade reagent with 1 5m/ml DAPI (final concentration 5µg/mL, can be stored at Room temperature).

Place 10µL solution in small droplets on the slides.

Place coverslip and press down gently to remove air pockets without disturbing the sample area.

Seal edges with clear nail polish.

Samples can now be stored at -20°C in covered container indefinitely.