TITLE: PhageFISH for DIG-labelled bacterial probes
AUTHORS: Line Jensen Ostenfeld, Saria Otani

[DESCRIPTION]
This protocol details about PhageFISH for DIG-labelled bacterial probes.

[STEPS]
SECTION: Fix liquid samples to glass slides
1. Place liquid sample in a 30-50 µL droplet on poly-L-lysine coated slide.

SECTION: Fix liquid samples to glass slides
2. Dry in warm incubator for approx. 30 min or until the droplet has dried out.

SECTION: Fix liquid samples to glass slides
3. OPTIONAL: if sample is very dilute add several droplets and repeat drying procedure.
SECTION: Fix liquid samples to glass slides
4. Add 1% paraformaldehyde to cover the sample area.
SECTION: Fix liquid samples to glass slides
5. Incubate at Room temperature for 60 min.

SECTION: Fix liquid samples to glass slides
6. Aspirate the paraformaldehyde off.
SECTION: Fix liquid samples to glass slides
7. Rinse samples in PBS for 1 min.

SECTION: Fix faecal samples to glass slides
8. Mix a small faecal sample with 10-20 µL PBS (1X) and vortex thoroughly.

SECTION: Fix faecal samples to glass slides
9. Allow suspension to settle for 5 min.

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

SECTION: Fix faecal samples to glass slides
11. Smear the droplet over the slide using a cover slip.
SECTION: Fix faecal samples to glass slides
12. Allow the sample to dry – this should not take more than 10 min.

SECTION: Fix faecal samples to glass slides
13. Overlay the slides with 1% paraformaldehyde. Ensure the whole sample area is covered (approx. 1 mL).

SECTION: Fix faecal samples to glass slides
14. Incubate for 60 min at Room temperature or   at 4 °C.

SECTION: Fix faecal samples to glass slides
15. Aspirate off excess paraformaldehyde.
SECTION: Fix faecal samples to glass slides
16. Wash in PBS for 1 min.
 


SECTION: Permeabilise cells
17. Add lysozyme to permeabilisation buffer.
SECTION: Permeabilise cells
18. Overlay samples with permeabilisation buffer.
SECTION: Permeabilise cells
19. Incubate on ice for 60 min.

SECTION: Permeabilise cells
20. Wash samples in PBS for 5 min.

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

SECTION: Inactivate peroxidases
22. Incubate samples in 0.01 Molarity (M) HCl for 10 min.

SECTION: Inactivate peroxidases
23. Wash samples in PBS for 5 min.

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

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

SECTION: Inactivate peroxidases
26. Allow slides to dry on blotting paper or filter paper.
SECTION: rRNA hybridisation of DIG-labelled probes
27. Place filters in a petri dish and spot up to 100 µL hybridisation buffer to cover the filters.
SECTION: rRNA hybridisation of DIG-labelled probes
28. Transfer to a humidity chamber with hybridisation buffer soaked paper towels.
SECTION: rRNA hybridisation of DIG-labelled probes
29. Incubate for 60 min at hybridisation temperature______.

SECTION: rRNA hybridisation of DIG-labelled probes
30. Mix 1 mL gene hybridisation buffer with 1 µL of each probe. Vortex to mix.

SECTION: rRNA hybridisation of DIG-labelled probes
31. Place one droplet of 30-100 µL probe mix on a petri dish for each filter.

SECTION: rRNA hybridisation of DIG-labelled probes
32. Place the filters face down in the probe mix droplets.
SECTION: rRNA hybridisation of DIG-labelled probes
33. Place the dish back in the humidity chamber and incubate for 60 min at 85 °C.

SECTION: rRNA hybridisation of DIG-labelled probes
34. Immediately place the humidity chamber at hybridisation temperature 60 min.
SECTION: rRNA hybridisation of DIG-labelled probes
35. Wash filters.
SECTION: rRNA hybridisation of DIG-labelled probes
36. Wash filters.
SECTION: rRNA hybridisation of DIG-labelled probes
37. Wash filters in PBS for 1 min.
SECTION: Antibody binding
38. Place filters in a petri dish and add antibody blocking solution to cover the filters. Incubate for 30 min.

SECTION: Antibody binding
39. Move filters to antibody binding solution and incubate for 90 min.
SECTION: Antibody binding
40. Wash filters.
SECTION: CARD amplification
41. Mix 1 mL amplification buffer with 10 µL H2O2 and 2 µL Alexa tyramides (488). Vortex to mix. 

SECTION: CARD amplification
42. Place filters in a petri dish and cover with probe mix by spotting droplets of 30-100 µL.
SECTION: CARD amplification
43. Wash filters.

SECTION: CARD amplification
44. Wash filters in sterile water for 1 min.

SECTION: CARD amplification
45. Wash filters in 96% ethanol for 1 min.

SECTION: Remove RNases
46. Add 10.8 mL sterile water, 1.2 mL Tris-HCl (1M, pH 8), 15 µL RNase I, and 30 µL RNase A to a 15ml falcon tube.
SECTION: Remove RNases
47. Place filters in the RNase solution and incubate for 60 min at 37 °C.

SECTION: Remove RNases
48. Wash filters in PBS for 5 min.

SECTION: Remove RNases
49. Repeat wash.
SECTION: Remove RNases
50. Wash filters in sterile water for 1 min.

SECTION: Gene hybridisation
51. Cover samples with hybridisation buffer.
SECTION: Gene hybridisation
52. Transfer to a humidity chamber with formamide soaked paper towels at the corresponding concentration.
SECTION: Gene hybridisation
53. Incubate for 60 min at hybridisation temperature (approx. 46 °C).

SECTION: Gene hybridisation
54. Mix 1 mL gene hybridisation buffer with 1 µL of each probe. Vortex to mix.

SECTION: Gene hybridisation
55. Cover the samples with the hybridisation buffer-probe mix.
SECTION: Gene hybridisation
56. Place the dish back in the humidity chamber and incubate for 60 min at 85 °C.

SECTION: Gene hybridisation
57. Immediately place the humidity chamber at hybridisation temperature 60 min.
 


SECTION: Gene hybridisation
58. Wash filters.
SECTION: Gene hybridisation
59. Wash filters.
SECTION: Gene hybridisation
60. Wash filters in PBS for 1 min.

SECTION: Antibody binding
61. Place filters in a petri dish and add antibody-blocking solution to cover the filters. Incubate for 30 min.

SECTION: Antibody binding
62. Move filters to antibody binding solution and incubate for 90 min.

SECTION: Antibody binding
63. Wash filters.
SECTION: CARD amplification
64. Mix 1 mL amplification buffer with 10 µL H2O2 and 2 µL Alexa tyramides (594). Vortex to mix. 
SECTION: CARD amplification
65. Place filters in a petri dish and cover with probe mix by spotting droplets of 30-100 µL. Incubate at 37 °C for 45 min.

SECTION: CARD amplification
66. Wash filters.
SECTION: CARD amplification
67. Wash filters in sterile water for 1 min.

SECTION: CARD amplification
68. Wash filters in 96% ethanol for 1 min.
 


SECTION: Staining
69. Mix 1 mL SlowFade Gold with 1 µL 5 mg/mL DAPI dye. 

SECTION: Staining
70. Apply 5-10 µL mix in droplets to each slide.

SECTION: Staining
71. Apply coverglass and carefully press down to seal sample with minimal air bubbles.
SECTION: Staining
72. Seal with clear nail polish on all edges of the sample.
SECTION: Staining
73. Allow to cure completely.
SECTION: Staining
74. Store at -20 °C.

SECTION: rRNA hybridisation of DIG-labelled probes
35.1. Wash filters in gene washing buffer I 1 min. (1/3)

SECTION: rRNA hybridisation of DIG-labelled probes
35.2. Wash filters in gene washing buffer I 1 min. (2/3)
SECTION: rRNA hybridisation of DIG-labelled probes
35.3. Wash filters in gene washing buffer I 1 min. (3/3)
SECTION: rRNA hybridisation of DIG-labelled probes
35.4. Wash filters in gene washing buffer I 30 min at 42 °C. 

SECTION: rRNA hybridisation of DIG-labelled probes
36.1. Wash filters in gene washing buffer II for 1 min. (1/3)
SECTION: rRNA hybridisation of DIG-labelled probes
36.2. Wash filters in gene washing buffer II for 1 min. (2/3)
SECTION: rRNA hybridisation of DIG-labelled probes
36.3. Wash filters in gene washing buffer II for 1 min. (3/3)
SECTION: rRNA hybridisation of DIG-labelled probes
36.4. Wash filters in gene washing buffer II for 90 min at 42 °C.

SECTION: Antibody binding
40.1. Wash filters in antibody washing solution for 1 min .
SECTION: Antibody binding
40.2. Wash filters in antibody washing solution for 10 min. (1/3)
SECTION: Antibody binding
40.3. Wash filters in antibody washing solution for 10 min. (2/3)
SECTION: Antibody binding
40.4. Wash filters in antibody washing solution for 10 min. (3/3)
SECTION: CARD amplification
43.1. Wash filters in PBS for 1 min
SECTION: CARD amplification
43.2. Wash filters in PBS for 5 min.

SECTION: CARD amplification
43.3. Wash filters in PBS for 10 min at 46 °C. (1/2)

SECTION: CARD amplification
43.4. Wash filters in PBS for 10 min at 46 °C. (2/2)

SECTION: Gene hybridisation
58.1. Wash filters in gene washing buffer I for 1 min. (1/3)

SECTION: Gene hybridisation
58.2. Wash filters in gene washing buffer I for 1 min. (2/3)

SECTION: Gene hybridisation
58.3. Wash filters in gene washing buffer I for 1 min. (3/3)

SECTION: Gene hybridisation
58.4. Wash filters in gene washing buffer I for 30 min at 42 °C.

SECTION: Gene hybridisation
59.1. Wash filters in gene washing buffer II for 1 min. (1/3)

SECTION: Gene hybridisation
59.2. Wash filters in gene washing buffer II for 1 min. (2/3)

SECTION: Gene hybridisation
59.3. Wash filters in gene washing buffer II for 1 min.(3/3)
SECTION: Gene hybridisation
59.4. Wash filters in gene washing buffer II for 90 min at 42 °C.

SECTION: Antibody binding
63.1. Wash filters in antibody washing solution for 1 min.

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

SECTION: Antibody binding
63.3. Wash filters in antibody washing solution for 10 min. (2/3)

SECTION: Antibody binding
63.4. Wash filters in antibody washing solution for 10 min. (3/3)

SECTION: CARD amplification
66.1. Wash filters in PBS for 1 min.

SECTION: CARD amplification
66.2. Wash filters in PBS for 5 min.

SECTION: CARD amplification
66.3. Wash filters in PBS for 10 min at 46 °C.

SECTION: CARD amplification
66.4. Wash filters in PBS for 10 min at 46 °C.