Feb 02, 2023

PhageFISH for DIG-labelled bacterial probes

PLOS One
Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3931pg25/v1
  • 1DTU
Saria Otani
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3931pg25/v1
Protocol Citation: Line Jensen Ostenfeld, Saria Otani 2023. PhageFISH for DIG-labelled bacterial probes. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3931pg25/v1
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 02, 2023
Protocol  Integer ID: 75844
Keywords: PhageFISH, DIG-labelled bacterial probes, phagefish for dig, bacterial probes this protocol detail, labelled bacterial probe, bacterial probe, phagefish, probe, dig
Funders Acknowledgements:
NovoNordisk
Grant ID: NNF16OC0021856
Abstract
This protocol details about PhageFISH for DIG-labelled bacterial probes.
Attachments
Icon for 627-1301.docx
627-1301.docx
32KB
Materials
Reagents

  • 1% paraformaldehyde
  • PBS
  • 0.01M HCl
  • sterile water
  • 96% ethanol
  • permeabilisation buffer
  • hybridisation buffer
  • gene washing buffer I
  • gene washing buffer II
  • amplification buffer
  • Alexa tyramides (488)
  • Tris-HCl
  • RNase I
  • RNase A
  • antibody-blocking solution
  • antibody binding solution
  • antibody washing solution
  • Alexa tyramides (594)
  • SlowFade Gold
  • DAPI dye
Fix liquid samples to glass slides
Place liquid sample in a 30-50 µL droplet on poly-L-lysine coated slide.

Dry in warm incubator for approx. 00:30:00 or until the droplet has dried out.

30m
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 01:00:00 .

1h
Aspirate the paraformaldehyde off.
Rinse samples in PBS for 00:01:00 .

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

Allow suspension to settle for 00:05:00 .

5m
Take 10 µL 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 00:10:00 .

10m
Overlay the slides with 1% paraformaldehyde. Ensure the whole sample area is covered (approx. 1 mL ).

Incubate for 01:00:00 at Room temperature or Overnight at 4 °C .

1h
Aspirate off excess paraformaldehyde.
Wash in PBS for 00:01:00 .
Note
FREEZING POINT


1m
Permeabilise cells
Add lysozyme to permeabilisation buffer.
Overlay samples with permeabilisation buffer.
Incubate On ice for 01:00:00 .

1h
Wash samples in PBS for 00:05:00 .

5m
Wash samples in sterile water for 00:01:00 .

1m
Inactivate peroxidases
Incubate samples in 0.01 Mass Percent HCl for 00:10:00 .

10m
Wash samples in PBS for 00:05:00 .

5m
Wash samples in sterile water for 00:01:00 .

1m
Wash samples in 96% ethanol for 00:01:00 .

1m
Allow slides to dry on blotting paper or filter paper.
rRNA hybridisation of DIG-labelled probes
Place filters in a petri dish and spot up to 100 µL hybridisation buffer to cover the filters.
Transfer to a humidity chamber with hybridisation buffer soaked paper towels.
Incubate for 01:00:00 at hybridisation temperature______.

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

Place one droplet of 30-100 µL 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 01:00:00 at 85 °C .

1h
Immediately place the humidity chamber at hybridisation temperature Overnight .
1h
Wash filters.
Wash filters in gene washing buffer I 00:01:00 . (1/3)

1m
Wash filters in gene washing buffer I 00:01:00 . (2/3)
1m
Wash filters in gene washing buffer I 00:01:00 . (3/3)
1m
Wash filters in gene washing buffer I 00:30:00 at 42 °C .

30m
Wash filters.
Wash filters in gene washing buffer II for 00:01:00 . (1/3)
1m
Wash filters in gene washing buffer II for 00:01:00 . (2/3)
1m
Wash filters in gene washing buffer II for 00:01:00 . (3/3)
1m
Wash filters in gene washing buffer II for 01:30:00 at 42 °C .

1h 30m
Wash filters in PBS for 00:01:00 .
1m
Antibody binding
Place filters in a petri dish and add antibody blocking solution to cover the filters. Incubate for 00:30:00 .

30m
Move filters to antibody binding solution and incubate for 01:30:00 .
1h 30m
Wash filters.
Wash filters in antibody washing solution for 00:01:00 .
1m
Wash filters in antibody washing solution for 00:10:00 . (1/3)
10m
Wash filters in antibody washing solution for 00:10:00 . (2/3)
10m
Wash filters in antibody washing solution for 00:10:00 . (3/3)
10m
CARD amplification
Mix 1 mL amplification buffer with 10 µL H2O2 and 2 µL Alexa tyramides (488). Vortex to mix.

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

Wash filters in PBS for 00:01:00
1m
Wash filters in PBS for 00:05:00 .

5m
Wash filters in PBS for 00:10:00 at 46 °C . (1/2)

10m
Wash filters in PBS for 00:10:00 at 46 °C . (2/2)

10m
Wash filters in sterile water for 00:01:00 .

1m
Wash filters in 96% ethanol for 00:01:00 .

1m
Remove RNases
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.
Place filters in the RNase solution and incubate for 01:00:00 at 37 °C .

1h
Wash filters in PBS for 00:05:00 .

5m
Repeat wash.
Wash filters in sterile water for 00:01:00 .

1m
Gene hybridisation
Cover samples with hybridisation buffer.
Transfer to a humidity chamber with formamide soaked paper towels at the corresponding concentration.
Incubate for 01:00:00 at hybridisation temperature (approx. 46 °C ).

1h
Mix 1 mL gene hybridisation buffer with 1 µL 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 01:00:00 at 85 °C .

1h
Immediately place the humidity chamber at hybridisation temperature Overnight .
Note
OVERNIGHT


1h
Wash filters.
Wash filters in gene washing buffer I for 00:01:00 . (1/3)

1m
Wash filters in gene washing buffer I for 00:01:00 . (2/3)

1m
Wash filters in gene washing buffer I for 00:01:00 . (3/3)

1m
Wash filters in gene washing buffer I for 00:30:00 at 42 °C .

30m
Wash filters.
Wash filters in gene washing buffer II for 00:01:00 . (1/3)

1m
Wash filters in gene washing buffer II for 00:01:00 . (2/3)

1m
Wash filters in gene washing buffer II for 00:01:00 .(3/3)
1m
Wash filters in gene washing buffer II for 01:30:00 at 42 °C .

1h 30m
Wash filters in PBS for 00:01:00 .

1m
Antibody binding
Place filters in a petri dish and add antibody-blocking solution to cover the filters. Incubate for 00:30:00 .

30m
Move filters to antibody binding solution and incubate for 01:30:00 .

1h 30m
Wash filters.
Wash filters in antibody washing solution for 00:01:00 .

1m
Wash filters in antibody washing solution for 00:10:00 . (1/3)

10m
Wash filters in antibody washing solution for 00:10:00 . (2/3)

10m
Wash filters in antibody washing solution for 00:10:00 . (3/3)

10m
CARD amplification
Mix 1 mL amplification buffer with 10 µL H2O2 and 2 µL Alexa tyramides (594). Vortex to mix.
Place filters in a petri dish and cover with probe mix by spotting droplets of 30-100 µL . Incubate at 37 °C for 00:45:00 .

45m
Wash filters.
Wash filters in PBS for 00:01:00 .

1m
Wash filters in PBS for 00:05:00 .

5m
Wash filters in PBS for 00:10:00 at 46 °C .

10m
Wash filters in PBS for 00:10:00 at 46 °C .

10m
Wash filters in sterile water for 00:01:00 .

1m
Wash filters in 96% ethanol for 00:01:00 .
Note
OPTIONAL FREEZING POINT


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

Apply 5-10 µL 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 .