Feb 20, 2023
HCR-fluorescent in situ hybridization (HCR-FISH) of gemmule-hatched freshwater sponges
- 1University of Denver
Protocol Citation: Scott Nichols 2023. HCR-fluorescent in situ hybridization (HCR-FISH) of gemmule-hatched freshwater sponges. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jwkdg2w/v1
Manuscript citation:
Colgren, J., Nichols, S.A. MRTF specifies a muscle-like contractile module in Porifera.Nat Commun13, 4134 (2022). https://doi.org/10.1038/s41467-022-31756-9
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: February 14, 2023
Last Modified: February 20, 2023
Protocol Integer ID: 76952
Funders Acknowledgement:
National Sciences Foundation
Grant ID: 2015608
Abstract
This HCR-FISH protocol using probes and amplifiers from Molecular Instruments is intended for gemmule-hatched freshwater sponges grown in 35 mm coverslip bottom cell-culture dishes with a 10 mm inner-well diameter.
Guidelines
We take reasonable measures to use RNAse-free reagents, but have successfully used this protocol with and without filter-tips.
Materials
Gemmule-hatched freshwater sponges
Sterile Filtered or autoclaved lakewater or store-bought spring water
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.
10x HF buffer (590mM NaCl; 6.7mM KCL; 7.6mM CaCl2; 2.4mM NaHCO) *note, make with RNAse-free molecular biology grade water
Cellbrite-Fix 640 (Biotium)
20% paraformaldehyde solution, methanol free (Electron Microscopy Solutions 15713-S)
SUPERase.in (AM2694 ThermoFisher)
100% Methanol
Reagent-grade (95%) Ethanol
1X PBS (RNAse-free)
1x PBS + 0.1% Tween (PTw) (RNAse-free)
Proteinase K, recombinant, PCR Grade,Solution (Sigma-Aldrich 03115828001)
2 mg/mL glycine (in PTw) (RNAse-free)
Probe Hybridization Buffer and Probe Wash Buffer (Molecular Instruments, HCR Buffer bundle #1 for whole-mount samples)
HCR probe sets with appropriate amplifiers and amplification buffer (Molecular Instruments)
Molecular Biology Grade water (RNAse/DNAse free)
20X SSC Buffer (RNAse-free) (e.g., Sigma-Aldrich S6639-1L)
Can use filter tips to minimize the risk of RNAse contamination
Thermalcycler
0.2 mL PCR tubes
Kim Wipes
100 mm petri dishes
Parafilm
Vectashield or other glycerol-based mounting medium with anti-fade properties
Hoechst (10 mg/mL stock solution)
Before start
Order probes and HCR-FISH kit from Molecular Instruments
Grow sponges in coverslip bottom dishes
Grow sponges in coverslip bottom dishes
Note
Briefly, add 3-4 mL of culture medium to a 35 mm, coverslip bottom culture dish with an inner well diameter of 10mm. Place 1-2 gemmules in the center of the inner well.
Grow the sponges for 168:00:00 ~ 1 week in the dark (this reduces the growth of Chlorella-like algal symbionts that autofluoresce (particularly in the far-red channel).
Note
Different gemmules develop at quite different rates. If you are interested in fully developed tissues, you should wait to fix tissues until you see well developed oscula, choanocyte chambers, and water canals.
1w
Membrane counterstaining and fixation
Membrane counterstaining and fixation
Remove culture medium from the outer well and inner well areas by aspiration or pipetting.
Note
Throughout this protocol, try to only remove liquid from the outer well area whenever possible, as pipetting and (particularly) aspiration from the inner well area can damage the sponge tissue. Always pipette slowly when adding and removing solutions.
Add 90-100 µL of 1:1000 Cellbrite-Fix 640 (Biotius) in lake/spring water to the inner well area for 00:15:00 at Room temperature in the dark.
Note
This step is optional, but useful because it allows you to see cell shape/boundaries to better understand to localization of your FISH signal. But, if you are interested in an easy-to-identify cell type such as choanocytes or archeocytes, a membrane counterstain may not be necessary.
Note
Depending upon your experimental design you may choose to use a different conjugate of Cellbrite-Fix other than 640 (e.g., 488). But, since Chlorella symbionts in cells autofluoresce strongly in the far-red channel it may make sense to counterstain in this channel to save "cleaner" channels for your FISH probes.
15m
To wash, it is not necessary to remove the staining solution. Instead, add 3 mL lake/spring water directly to the outer well area.
Immediately remove the wash by pipetting or aspiration from the outer well area.
Add 3 mL of cold 4% paraformaldehyde in 1/4x HF Buffer to the outer well area. Place at 4 °C Overnight in the dark.
Safety information
You should not breathe the fumes of Formaldehyde. Work inside a chemical fume hood.
Note
At this point, and in all subsequent steps, it is important to keep your samples shielded from light as much as possible to protect your Cellbrite signal
Treat with RNAse inhibitor
Treat with RNAse inhibitor
Remove 3mLs of fixative from the outer well area. It is not necessary to first remove the residual liquid in the inner well area around the sponge. (Dispose of formaldehyde-containing waste properly)
Add 4 mLs of 1/4x HF Buffer to the outer well area of the dish. Incubate 00:05:00
5m
Remove the wash from the outer well area, and repeat step 9.
Remove the wash from the outer and inner well areas (be very careful pipetting near the tissue).
Add 100µl of ¼ x HF buffer containing an RNAse inhibitor (1 U/µl final concentration) to the inner well. Incubate at Room temperature for 00:10:00 .
10m
Wash by adding 3 mL ¼ x HF Buffer to the outer well area for 00:03:00 at Room temperature .
3m
Remove wash by pipetting from the outer well area only, then repeat wash (step 13) once.
3m
Dehydrate
Dehydrate
Remove the wash buffer from the outer well area of the dish and add 2 mL of 30% Methanol diluted in ¼ x HF Buffer. Incubate 00:05:00 at Room temperature
Safety information
Work in a fume hood to avoid breathing methanol fumes, and dispose of waste properly.
5m
Remove 30% Methanol solution from the outer well area and add 2 mL of 70% Methanol diluted in ¼ x HF Buffer. Incubate 00:05:00 at Room temperature
5m
Remove 70% Methanol solution from the outer well area and add 3 mL of 100% Methanol. Incubate 00:05:00 at Room temperature
Note
This could be a stopping point if you want to parafilm the sample and store at -20 C overnight.
5m
Remove the Methanol from the outer well area and replace with 3 mL 100% Ethanol. Incubate 00:05:00 at Room temperature
5m
Rehydrate and Permeabilize
Rehydrate and Permeabilize
Remove the 100% Ethanol from the outer well area and replace with 2 mL of 70% Ethanol diluted in PTw (1x PBS + 0.1% Tween-20). Incubate 00:05:00 at Room temperature
5m
Remove the 70% Ethanol from the outer well area and replace with 2 mL of 30% Ethanol diluted in PTw. Incubate 00:05:00 at Room temperature
5m
Remove the 30% Ethanol from the outer well area and replace with 2 mL of 30% Ethanol diluted in PTw. Incubate 00:05:00 at Room temperature
5m
Remove 30% Ethanol from the outer well area and replace with 2 mL of PTw. Incubate 00:05:00 at Room temperature
5m
Repeat PTw wash, 1 time.
Proteinase K treatment
Proteinase K treatment
Remove the PTw from the outer well area and replace with 1 mL of 5 µg/mL Proteinase K (in PTw) for 00:01:30 at Room temperature
1m 30s
Remove the Proteinase K treatment from the inner and outer well area and replace with 2 mL of 2 mg/mL glycine (in PTw) for 00:05:00 at Room temperature
5m
Remove the glycine solution from the outer well area and add an additional 2 mL of 2 mg/mL glycine (in PTw) for 00:05:00 at Room temperature
5m
Remove the glycine solution from the outer well area and replace with 2 mL of PTw for 00:05:00 at Room temperature
5m
Remove the PTw from the outer well area and add an additional 2 mL of PTw for 00:05:00 at Room temperature
5m
Post-fixation and Wash
Post-fixation and Wash
Remove PTw from outer well area and replace with 3 mL of 4% paraformaldhyde in PTw. Incubate for 01:00:00 at Room temperature .
Safety information
Formaldehyde fumes are toxin. Work in a chemical fume hood, and dispose of waste appropriately.
1h
Remove fixative from the outer well area, and replace with 3 mL of PTw. Incubate for 00:10:00 at Room temperature .
10m
Remove PTw from outer well area and add an addition 3 mL PTw to the outer well area. Incubate for 00:10:00 at Room temperature .
10m
Remove PTw from outer well area and replace with 3 mL of 2x SSC. Incubate for 00:10:00 at Room temperature
10m
Remove 2x SSC from the outer well area and replace with 3 mL of 2x SSC. Incubate for 00:10:00 at Room temperature
10m
Prehybridization
Prehybridization
Bring 30% hybridization buffer (HB) to Room temperature
Safety information
Contains formamide. Use inside a chemical fume hood and dispose of waste properly.
Remove 2x SSC from outer and inner well area.
Add 1 mL HB to the dish. Incubate for 00:10:00 at Room temperature
10m
Remove HB from outer well area and replace with 1 mL HB. Incubate for 00:30:00 at 37 °C
30m
Probe hybridization
Probe hybridization
During Prehybridization, prepare probe solutions.
Note
Molecular Instruments provides probes at 1 µM, with the recommendation to use 0.5 µl in a 100 µl final volume. We find that this concentration works well, but for characterizing a new probe we often also test a higher concentration, such as 5 µl probe in a 100 µl final volume.
If using more than one probe, combine both in the same 100 µl volume per sample. (e.g., 1 µl probe 1 + 1 µl probe 2 + 98 µl HB)
After 30 min incubation in HB (minimum), remove HB from the outer and inner wells, then add 100 µL probe solution to the inner well area only.
Incubate on a very gently rocking platform at 37 °C Overnight .
Note
It is important that the rocking platform VERY slow and that the angle of incline is not so great that the probe solution drains out of the inner well. It is probably fine to leave the sample stationary, but we haven't tested this.
Washes
Washes
Heat 30% Probe Wash Buffer (PWB) to 37 °C (4 mL per sample).
Add 1 mL preheated 30% PWB to the outer well area (it is not necessary to first remove the probe solution from the inner well area). Incubate at 37 °C for 00:05:00
5m
Remove PWB from outer well area and replace with 1 mL 30% PWB to outer well area. Incubate at 37 °C for 00:05:00
5m
Remove PWB from outer well area and replace with 1 mL 30% PWB to outer well area. Incubate at 37 °C for 00:05:00
5m
Remove PWB from outer well area and replace with 1 mL 30% PWB to outer well area. Incubate at 37 °C for 00:05:00
5m
Remove PWB from outer well area and replace with 1 mL 2x SSC. Incubate at Room temperature for 00:05:00
5m
Remove 2x SSC from outer well area and replace with 1 mL 2x SSC. Incubate at Room temperature for 00:05:00
5m
Prepare Amplifiers
Prepare Amplifiers
Bring Amplification Buffer (AB) to Room temperature
Note
If you are preparing more than one sample, calculate for the # of samples + 1 so that you have plenty of amplifier solution after accounting for pipetting error.
Add 6 pmol (2 µL of a 3 µM stock) of the appropriate amplifier hairpin 1 to 0.2 mL PCR tube.
Add 6 pmol (2 µL of a 3 µM stock) of the appropriate amplifier hairpin 2 to a 0.2 mL PCR tube.
Add amplifier samples to preheated 95 °C thermal cycler for exactly 00:01:30 .
1m 30s
Remove samples from thermal cycler and place in the dark at Room temperature for 00:30:00
30m
Combine 2 µL of hairpin 1 and 2 µL of hairpin 2 (and additional hairpins if multiplexing) into 100 µL final volume of AB at Room temperature .
Note
This assumes 1 sample. If doing more samples, just scale the volume of hairpins and AB needed. Also, you will combine amplifiers with different fluorophores into the same tube of AB at this point if you are multiplexing.
Amplification
Amplification
Remove 2x SSC from the outer and inner well area, then add 100 µL of AB to the inner well. Incubate for 00:05:00 at Room temperature
5m
Remove AB from the inner well area and replace with 100 µL of AB. Incubate for 00:05:00 at Room temperature
5m
Remove AB from the inner well area and add 100 µL of the hairpin solution from step 53.
Place up to 3 samples into a 100 mm Petri dish. Add water-soaked kimwipes to the space between two samples and close the lid. Seal the Petri dish with a strip of parafilm, being very careful not to spill the amplifier solution out of the inner well.
Incubate in the dark at Room temperature Overnight .
Counterstaining, Mounting, and Imaging
Counterstaining, Mounting, and Imaging
30m
30m
Add 2 mL 2x SSC to the outer well area (it is not necessary to first remove the amplifier hairpin solution). Incubate 00:05:00 at Room temperature
5m
Remove 2x SSC from the outer well area and replace with 2 mL 2x SSC. Incubate 00:05:00 at Room temperature .
5m
Remove 2x SSC and add 1 mL PBS containing 1:100 Hoechst solution. Incubate 00:20:00 at Room temperature .
20m
Remove PBS/Hoechst solution and replace with 2 mL PBS.
Remove PBS from outer and inner well areas.
Add 100 µL Vectashield (or other antifade mounting medium) to the inner well area. Store at 4 °C until ready to image.