TITLE: PhageFISH detailed protocol
AUTHORS: Line Jensen Ostenfeld, Saria Otani

[DESCRIPTION]
This protocol details about PhageFISh protocol.

[BEFORE_START]
Prepare buffers (see Preparation of Buffers for PhageFISH protocol).

[GUIDELINES]

Controls to consider: 

Faecal sample with no target for the phage probe

Timeframe: 
 
 
   
 

[STEPS]
SECTION: Fix faecal samples to glass slides
1. Mix a loopful faecal sample with 10-20 µL PBS (1X) and vortex thoroughly.
SECTION: Fix faecal samples to glass slides
2. Allow suspension to settle for 5 min to avoid large debris.

SECTION: Fix faecal samples to glass slides
3. Take 10 µL of the supernatant and place on coated glass slide.

SECTION: Fix faecal samples to glass slides
4. Smear the droplet thinly over the slide using a cover slip.
 

SECTION: Fix faecal samples to glass slides
5. Allow the sample to dry – this should not take more than 10 min.
 


SECTION: Fix faecal samples to glass slides
6. Work in fume hood. Overlay the slides with 1% paraformaldehyde (PFA). Ensure the whole sample area is covered (approx. 1 mL).

SECTION: Fix faecal samples to glass slides
7. Incubate for 60 min at Room temperature in the fume hood. 
 


SECTION: Fix faecal samples to glass slides
8. Aspirate off excess PFA.
SECTION: Fix faecal samples to glass slides
9. Wash in PBS for 1 min .
 


SECTION: Permeabilise cells
10. Add lysozyme to permeabilisation buffer.
SECTION: Permeabilise cells
11. Overlay samples with permeabilisation buffer.
SECTION: Permeabilise cells
12. Incubate on ice for 60 min.

SECTION: Permeabilise cells
13. Discard permeabilisation buffer.
SECTION: Permeabilise cells
14. Wash samples in PBS for 5 min.

SECTION: Permeabilise cells
15. Wash samples in sterile water for 1 min.

SECTION: Inactivate peroxidases
16. Incubate samples in 0.01 Molarity (M) HCl for 10 min.
SECTION: Inactivate peroxidases
17. Wash samples in PBS for 5 min.

SECTION: Inactivate peroxidases
18. Wash samples in sterile water for 1 min.

SECTION: Inactivate peroxidases
19. Wash samples in 96% ethanol for 1 min.

SECTION: Inactivate peroxidases
20. Allow slides to dry on blotting paper or filter paper.
 

SECTION: Cy-labelled probe hybridisation (16S rRNA probes)
21. 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. 
SECTION: Cy-labelled probe hybridisation (16S rRNA probes)
22. Overlay samples with hybridisation buffer-probe mix at 0.5 ng/µl of each probe and close humidity chamber.
SECTION: Cy-labelled probe hybridisation (16S rRNA probes)
23. Incubate at 46 °C for 180 min.
SECTION: Cy-labelled probe hybridisation (16S rRNA probes)
24. Prepare the washing buffer – heat to 48 °C.
SECTION: Cy-labelled probe hybridisation (16S rRNA probes)
25. Work in fume hood. Overlay the samples with washing buffer and incubate for 15 min at 48 °C (in humidity chamber to avoid formamide fumes).

SECTION: Cy-labelled probe hybridisation (16S rRNA probes)
26. Wash samples in sterile water.
SECTION: Cy-labelled probe hybridisation (16S rRNA probes)
27. Allow samples to dry.
 

SECTION: Phage probe hybridisation
28. 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. 
SECTION: Phage probe hybridisation
29. Overlay samples with hybridisation buffer (no probes!) and close humidity chamber (500 µL per slide).

SECTION: Phage probe hybridisation
30. Incubate for 60 min at 46 °C.

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

SECTION: Phage probe hybridisation
32. Place the dish back in the humidity chamber and incubate for 60 min at 85 °C.

SECTION: Phage probe hybridisation
33. Immediately place the humidity chamber at hybridisation temperature 60 min.

SECTION: Phage probe hybridisation
34. Wash slides.
SECTION: Phage probe hybridisation
35. Wash slides.
36. Wash slides in PBS for 1 min.

SECTION: Antibody binding
37. Cover slides with antibody-blocking solution. Incubate for 30 min.

SECTION: Antibody binding
38. Discard antibody-blocking solution and cover with antibody binding solution. Incubate for 90 min.

SECTION: Antibody binding
39. Wash slides.
SECTION: CARD amplification
40. Mix 1 mL amplification buffer with 10 µL H2O2 and 2 µL Alexa tyramides (488). Vortex to mix. 

SECTION: CARD amplification
41. Cover slides with CARD buffer-tyramide mix (approx. 500 µL per slide). Incubate at 37 °C for 45 min.

SECTION: CARD amplification
42. Wash slides.
SECTION: CARD amplification
43. Wash slides in sterile water for 1 min.

SECTION: CARD amplification
44. Wash slides in 96% ethanol for 1 min.
 


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

SECTION: Staining and embedding
46. Place 10 µL solution in small droplets on the slides.

SECTION: Staining and embedding
47. Place coverslip and press down gently to remove air pockets without disturbing the sample area.
SECTION: Staining and embedding
48. Seal edges with clear nail polish.
SECTION: Staining and embedding
49. Samples can now be stored at -20 °C in covered container indefinitely.

SECTION: Phage probe hybridisation
34.1. Wash slides in gene washing buffer I for 1 min. (1/3)

SECTION: Phage probe hybridisation
34.2. Wash slides in gene washing buffer I for 1 min. (2/3)
SECTION: Phage probe hybridisation
34.3. Wash slides in gene washing buffer I for 1 min. (3/3)
SECTION: Phage probe hybridisation
34.4. Wash slides in gene washing buffer I for 30 min at 42 °C.

SECTION: Phage probe hybridisation
35.1. Wash slides in gene washing buffer II for 1 min. (1/3)

35.2. Wash slides in gene washing buffer II for 1 min. (2/3)
35.3. Wash slides in gene washing buffer II for 1 min. (3/3)
35.4. Wash slides in gene washing buffer II for 90 min at 42 °C.

SECTION: Antibody binding
39.1. Wash slides in antibody washing solution for 1 min.

SECTION: Antibody binding
39.2. Wash slides in antibody washing solution for 10 min.  (1/3)

SECTION: Antibody binding
39.3. Wash slides in antibody washing solution for 10 min.  (2/3)
SECTION: Antibody binding
39.4. Wash slides in antibody washing solution for 10 min.  (3/3)

SECTION: CARD amplification
42.1. Wash slides in PBS for 1 min.

SECTION: CARD amplification
42.2. Wash slides in PBS for 5 min.

SECTION: CARD amplification
42.3. Wash slides in PBS for 10 min at 46 °C.

SECTION: CARD amplification
42.4. Wash slides in PBS for 10 min at 46 °C.