License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Protocol status: Working We use this protocol and it's working
Created: January 25, 2023
Last Modified: February 01, 2023
Protocol Integer ID: 75844
Keywords: PhageFISH, DIG-labelled bacterial probes
Funders Acknowledgement: NovoNordisk
Grant ID: NNF16OC0021856
Fix liquid samples to glass slides
Fix liquid samples to glass slides
1 Place liquid sample in a Amount 30-50 µL droplet on poly-L-lysine coated slide.
2 Dry in warm incubator for approx. Duration 00:30:00 or until the droplet has dried out.
3 OPTIONAL: if sample is very dilute add several droplets and repeat drying procedure.
4 Add 1% paraformaldehyde to cover the sample area.
5 Incubate at Temperature Room temperature for Duration 01:00:00 .
6 Aspirate the paraformaldehyde off.
7 Rinse samples in PBS for Duration 00:01:00 .
Fix faecal samples to glass slides
Fix faecal samples to glass slides
8 Mix a small faecal sample with Amount 10-20 µL PBS (1X) and vortex thoroughly.
9 Allow suspension to settle for Duration 00:05:00 .
10 Take Amount 10 µL of the supernatant and place on coated glass slide.
11 Smear the droplet over the slide using a cover slip.
12 Allow the sample to dry – this should not take more than Duration 00:10:00 .
13 Overlay the slides with 1% paraformaldehyde. Ensure the whole sample area is covered (approx. Amount 1 mL ).
14 Incubate for Duration 01:00:00 at Temperature Room temperature or Duration Overnight at Temperature 4 °C .
15 Aspirate off excess paraformaldehyde.
16 Wash in PBS for Duration 00:01:00 .
Permeabilise cells
Permeabilise cells
17 Add lysozyme to permeabilisation buffer.
18 Overlay samples with permeabilisation buffer.
19 Incubate Temperature On ice for Duration 01:00:00 .
20 Wash samples in PBS for Duration 00:05:00 .
21 Wash samples in sterile water for Duration 00:01:00 .
Inactivate peroxidases
Inactivate peroxidases
22 Incubate samples in Concentration 0.01 Molarity (M) HCl for Duration 00:10:00 .
23 Wash samples in PBS for Duration 00:05:00 .
24 Wash samples in sterile water for Duration 00:01:00 .
25 Wash samples in 96% ethanol for Duration 00:01:00 .
26 Allow slides to dry on blotting paper or filter paper.
rRNA hybridisation of DIG-labelled probes
rRNA hybridisation of DIG-labelled probes
27 Place filters in a petri dish and spot up to Amount 100 µL hybridisation buffer to cover the filters.
28 Transfer to a humidity chamber with hybridisation buffer soaked paper towels.
29 Incubate for Duration 01:00:00 at hybridisation temperature______.
30 Mix Amount 1 mL gene hybridisation buffer with Amount 1 µL of each probe. Vortex to mix.
31 Place one droplet of Amount 30-100 µL probe mix on a petri dish for each filter.
32 Place the filters face down in the probe mix droplets.
33 Place the dish back in the humidity chamber and incubate for Duration 01:00:00 at Temperature 85 °C .
34 Immediately place the humidity chamber at hybridisation temperature Duration Overnight .
35.1 Wash filters in gene washing buffer I Duration 00:01:00 . (1/3)
35.2 Wash filters in gene washing buffer I Duration 00:01:00 . (2/3)
35.3 Wash filters in gene washing buffer I Duration 00:01:00 . (3/3)
35.4 Wash filters in gene washing buffer I Duration 00:30:00 at Temperature 42 °C .
36.1 Wash filters in gene washing buffer II for Duration 00:01:00 . (1/3)
36.2 Wash filters in gene washing buffer II for Duration 00:01:00 . (2/3)
36.3 Wash filters in gene washing buffer II for Duration 00:01:00 . (3/3)
36.4 Wash filters in gene washing buffer II for Duration 01:30:00 at Temperature 42 °C .
37 Wash filters in PBS for Duration 00:01:00 .
Antibody binding
Antibody binding
38 Place filters in a petri dish and add antibody blocking solution to cover the filters. Incubate for Duration 00:30:00 .
39 Move filters to antibody binding solution and incubate for Duration 01:30:00 .
40.1 Wash filters in antibody washing solution for Duration 00:01:00 .
40.2 Wash filters in antibody washing solution for Duration 00:10:00 . (1/3)
40.3 Wash filters in antibody washing solution for Duration 00:10:00 . (2/3)
40.4 Wash filters in antibody washing solution for Duration 00:10:00 . (3/3)
CARD amplification
CARD amplification
41 Mix Amount 1 mL amplification buffer with Amount 10 µL H 2 O 2 and Amount 2 µL Alexa tyramides (488). Vortex to mix.
42 Place filters in a petri dish and cover with probe mix by spotting droplets of Amount 30-100 µL .
43.1 Wash filters in PBS for Duration 00:01:00
43.2 Wash filters in PBS for Duration 00:05:00 .
43.3 Wash filters in PBS for Duration 00:10:00 at Temperature 46 °C . (1/2)
43.4 Wash filters in PBS for Duration 00:10:00 at Temperature 46 °C . (2/2)
44 Wash filters in sterile water for Duration 00:01:00 .
45 Wash filters in 96% ethanol for Duration 00:01:00 .
Remove RNases
Remove RNases
46 Add Amount 10.8 mL sterile water, Amount 1.2 mL Tris-HCl (1M, pH 8), Amount 15 µL RNase I, and Amount 30 µL RNase A to a 15ml falcon tube.
47 Place filters in the RNase solution and incubate for Duration 01:00:00 at Temperature 37 °C .
48 Wash filters in PBS for Duration 00:05:00 .
50 Wash filters in sterile water for Duration 00:01:00 .
Gene hybridisation
Gene hybridisation
51 Cover samples with hybridisation buffer.
52 Transfer to a humidity chamber with formamide soaked paper towels at the corresponding concentration.
53 Incubate for Duration 01:00:00 at hybridisation temperature (approx. Temperature 46 °C ).
54 Mix Amount 1 mL gene hybridisation buffer with Amount 1 µL of each probe. Vortex to mix.
55 Cover the samples with the hybridisation buffer-probe mix.
56 Place the dish back in the humidity chamber and incubate for Duration 01:00:00 at Temperature 85 °C .
57 Immediately place the humidity chamber at hybridisation temperature Duration Overnight .
58.1 Wash filters in gene washing buffer I for Duration 00:01:00 . (1/3)
58.2 Wash filters in gene washing buffer I for Duration 00:01:00 . (2/3)
58.3 Wash filters in gene washing buffer I for Duration 00:01:00 . (3/3)
58.4 Wash filters in gene washing buffer I for Duration 00:30:00 at Temperature 42 °C .
59.1 Wash filters in gene washing buffer II for Duration 00:01:00 . (1/3)
59.2 Wash filters in gene washing buffer II for Duration 00:01:00 . (2/3)
59.3 Wash filters in gene washing buffer II for Duration 00:01:00 .(3/3)
59.4 Wash filters in gene washing buffer II for Duration 01:30:00 at Temperature 42 °C .
60 Wash filters in PBS for Duration 00:01:00 .
Antibody binding
Antibody binding
61 Place filters in a petri dish and add antibody-blocking solution to cover the filters. Incubate for Duration 00:30:00 .
62 Move filters to antibody binding solution and incubate for Duration 01:30:00 .
63.1 Wash filters in antibody washing solution for Duration 00:01:00 .
63.2 Wash filters in antibody washing solution for Duration 00:10:00 . (1/3)
63.3 Wash filters in antibody washing solution for Duration 00:10:00 . (2/3)
63.4 Wash filters in antibody washing solution for Duration 00:10:00 . (3/3)
CARD amplification
CARD amplification
64 Mix Amount 1 mL amplification buffer with Amount 10 µL H 2 O 2 and Amount 2 µL Alexa tyramides (594). Vortex to mix.
65 Place filters in a petri dish and cover with probe mix by spotting droplets of Amount 30-100 µL . Incubate at Temperature 37 °C for Duration 00:45:00 .
66.1 Wash filters in PBS for Duration 00:01:00 .
66.2 Wash filters in PBS for Duration 00:05:00 .
66.3 Wash filters in PBS for Duration 00:10:00 at Temperature 46 °C .
66.4 Wash filters in PBS for Duration 00:10:00 at Temperature 46 °C .
67 Wash filters in sterile water for Duration 00:01:00 .
68 Wash filters in 96% ethanol for Duration 00:01:00 .
69 Mix Amount 1 mL SlowFade Gold with Amount 1 µL Amount 5 mg/mL DAPI dye.
70 Apply Amount 5-10 µL mix in droplets to each slide.
71 Apply coverglass and carefully press down to seal sample with minimal air bubbles.
72 Seal with clear nail polish on all edges of the sample.
73 Allow to cure completely.
74 Store at Temperature -20 °C .