Sep 02, 2024
Bacterial fluorescent in situ hybridization (FISH) in Ephydatia muelleri
- Haley Womack1,
- Gwendolyn Geiger1,
- Scott Nichols1
- 1University of Denver
Protocol Citation: Haley Womack, Gwendolyn Geiger, Scott Nichols 2024. Bacterial fluorescent in situ hybridization (FISH) in Ephydatia muelleri. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49w52go5/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: June 11, 2024
Last Modified: September 02, 2024
Protocol Integer ID: 101617
Keywords: bacterial fluorescent in situ hybridization, eubacteria fish probe, bacterial fluorescent, hatched freshwater sponge, ephydatia muelleri this protocol, ephydatia muelleri, freshwater sponge, bacteria, situ hybridization, fish
Funders Acknowledgements:
National Science Foundation
Grant ID: 2015608
Abstract
This protocol is used to visualize bacteria in and around gemmule-hatched freshwater sponges using a Eubacteria FISH probe.
Materials
Freshwater sponge gemmules.
35 mm coverslip-bottom dishes with a 10 mm inner well diameter (Mattek #P35G-0-10-C). Note: you can use a different coverslip thickness, but the diameter of the inner well works with the volumes suggested in this protocol.
Fixative [4% formaldehyde (F8775-25ML Millepore) in 95% reagent alcohol].
PBS-Tw [1x PBS (41620012 bioPLUS) containing 0.1% Tween-20 (P1379-500ML Sigma-Aldrich)].
Formamide, Deionized (0606-950mL Amresco).
SSC (S24022-1000.0 RPI).
Eub338 probe (Amann et al., 1995. Microbiological reviews.).
Hybridization Buffer (Found in Molecular Instruments HCR‱ buffers pack in the HCR‱ RNA-FISH bundle).
Hoechst 33342 stock solution [10mg/mL] (40046 Biotium).
Mounting medium [either Vectashield (H-1000 Vector Laboratories) or equivalent].
Troubleshooting
Safety warnings
Work with formaldehyde and formamide (also present in Hybridization Buffer) in a chemical fume hood and dispose of waste appropriately.
Grow sponges
Add 3-4 mL mL of spring water to a 35mm coverslip bottom dish and place 1-3 gemmules into the center of the inner well.
Let grow in the dark (to reduce autofluorescence of algae) at Room temperature for about 168:00:00 (1 week) or until tissue appears developed.
1w
Day One
6h
Make fixative of 4% formaldehyde in 95% EtOH
150 µL per sample
Safety information
Do not breathe in fumes from formaldehyde, handle this chemical in a fume hood.
Day One
6h
Remove water from the outer well area, add 150 µL of fixative to the sponge, and let sit at room temperature for 04:00:00
Note
When pipetting from the wells, do so slowly from the outer well to not disturb sponge tissue. Also pipette slowly when adding solutions to the inner well.
4h
Closer to the end of the incubation, prepare a 0.1% solution of Tween-20 in 1X PBS (now referred to as PBS-Tw)
12 mL per sample
Prepare 10% formamide in 2X SSC
2 mL per sample
Safety information
Do not breathe in fumes from formamide, handle this chemical in a fume hood.
Remove the fixative from the outer well area and replace with 4 mL of PBS-Tw to each well
Remove the PBS-Tw and repeat the wash two more times
Add 2 mL of freshly prepared 10% formamide in 2X SSC to each sample and incubate at 37 °C for 02:00:00
2h
Prepare 1:100 eub338 probe [1µM final concentration] in Hybridization Buffer (Molecular Instruments) and heat to 37℃
Note
After this point, keep samples out of the light to protect the conjugated FITC probe
Remove the 10% formamide in 2X SSC from the samples and add 80-100 µL of probe mixture to the inner well, being careful to leave the samples flat
Place lid on sample and leave in humid chamber Overnight at 37℃
Note
It may be helpful to place damp KimWipes in between samples in a Tupperware container to keep the samples from drying out
Day Two
50m
Prepare 10% formamide in 2X SSC
3 mL per sample
Prepare 2X SSC
3 mL per sample
Prepare 0.2X SSC
12 mL per sample
Warm all solutions to 37 °C in a water bath
Safety information
Do not breathe in fumes from formamide, handle this chemical in a fume hood.
Remove probe mixture from samples and add 3 mL of pre-warmed 10% formamide in 2X SSC. Incubate for 00:10:00 at 37 °C
10m
Remove the 10% formamide in 2X SSC from the wells and replace with 3 mL of pre-warmed 2X SSC. Incubate for 00:10:00 at 37 °C
10m
Remove the 2X SSC from the wells and replace with 3 mL of pre-warmed 0.2X SSC. Incubate for 00:15:00 at 37 °C
15m
Repeat step 15 two more times, for a total of three washes
30m
Prepare a 1:200 dilution of Hoechst (stock concentration of 10mg/mL) in 0.2X SSC
100 µL per sample
Remove 0.2X SSC from wells and add about 100 µL of Hoechst solution to each inner well. Incubate for 00:15:00 at Room temperature
15m
Wash samples with 3mL of 0.2X SSC
Remove wash and add mounting medium
Samples are ready to be imaged, or can be left in the dark at 4 °C for preferably up to a week