HCR-fluorescent in situ hybridization (HCR-FISH) of gemmule-hatched freshwater sponges
Scott Nichols
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.
Before start
Order probes and HCR-FISH kit from Molecular Instruments
Steps
Grow sponges in coverslip bottom dishes
3-4mL 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 168h 0m 0s in the dark (this reduces the growth of Chlorella-like algal symbionts that autofluoresce (particularly in the far-red channel).
Membrane counterstaining and fixation
Remove culture medium from the outer well and inner well areas by aspiration or pipetting.
Add 90-100µL of 1:1000 Cellbrite-Fix 640 (Biotius) in lake/spring water to the inner well area for 0h 15m 0s at Room temperature in the dark.
To wash, it is not necessary to remove the staining solution. Instead, add 3mL lake/spring water directly to the outer well area.
Immediately remove the wash by pipetting or aspiration from the outer well area.
Add 3mL of cold 4% paraformaldehyde in 1/4x HF Buffer to the outer well area. Place at 4°C in the dark.
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 0h 5m 0s
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 0h 10m 0s .
Wash by adding 3mL ¼ x HF Buffer to the outer well area for 0h 3m 0s at Room temperature .
Remove wash by pipetting from the outer well area only, then repeat wash (step 13) once.
Dehydrate
Remove the wash buffer from the outer well area of the dish and add 2mL of 30% Methanol diluted in ¼ x HF Buffer. Incubate 0h 5m 0s at Room temperature
Remove 30% Methanol solution from the outer well area and add 2mL of 70% Methanol diluted in ¼ x HF Buffer. Incubate 0h 5m 0s at Room temperature
Remove 70% Methanol solution from the outer well area and add 3mL of 100% Methanol. Incubate 0h 5m 0s at Room temperature
Remove the Methanol from the outer well area and replace with 3mL 100% Ethanol. Incubate 0h 5m 0s at Room temperature
Rehydrate and Permeabilize
Remove the 100% Ethanol from the outer well area and replace with 2mL of 70% Ethanol diluted in PTw (1x PBS + 0.1% Tween-20). Incubate 0h 5m 0s at Room temperature
Remove the 70% Ethanol from the outer well area and replace with 2mL of 30% Ethanol diluted in PTw. Incubate 0h 5m 0s at Room temperature
Remove the 30% Ethanol from the outer well area and replace with 2mL of 30% Ethanol diluted in PTw. Incubate 0h 5m 0s at Room temperature
Remove 30% Ethanol from the outer well area and replace with 2mL of PTw. Incubate 0h 5m 0s at Room temperature
Repeat PTw wash, 1 time.
Proteinase K treatment
Remove the PTw from the outer well area and replace with 1mL of 5 µg/mL Proteinase K (in PTw) for 0h 1m 30s at Room temperature
Remove the Proteinase K treatment from the inner and outer well area and replace with 2mL of 2 mg/mL glycine (in PTw) for 0h 5m 0s at 5Room temperature
Remove the glycine solution from the outer well area and add an additional 2mL of 2 mg/mL glycine (in PTw) for 0h 5m 0s at 5Room temperature
Remove the glycine solution from the outer well area and replace with 2mL of PTw for 0h 5m 0s at 5Room temperature
Remove the PTw from the outer well area and add an additional 2mL of PTw for 0h 5m 0s at 5Room temperature
Post-fixation and Wash
Remove PTw from outer well area and replace with 3mL of 4% paraformaldhyde in PTw. Incubate for 1h 0m 0s at Room temperature .
Remove fixative from the outer well area, and replace with 3mL of PTw. Incubate for 0h 10m 0s at Room temperature .
Remove PTw from outer well area and add an addition 3mL PTw to the outer well area. Incubate for 0h 10m 0s at Room temperature .
Remove PTw from outer well area and replace with 3mL of 2x SSC. Incubate for 0h 10m 0s at Room temperature
Remove 2x SSC from the outer well area and replace with 3mL of 2x SSC. Incubate for 0h 10m 0s at Room temperature
Prehybridization
Bring 30% hybridization buffer (HB) to Room temperature
Remove 2x SSC from outer and inner well area.
Add 1mL HB to the dish. Incubate for 0h 10m 0s at Room temperature
Remove HB from outer well area and replace with 1mL HB. Incubate for 0h 30m 0s at 37°C
Probe hybridization
During Prehybridization, prepare probe solutions.
After 30 min incubation in HB (minimum), remove HB from the outer and inner wells, then add 100µL probe solution to the inner well area only.
Incubate on a very gently rocking platform at 37°C .
Washes
Heat 30% Probe Wash Buffer (PWB) to 37°C (4mL per sample).
Add 1mL 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 0h 5m 0s
Remove PWB from outer well area and replace with 1mL 30% PWB to outer well area. Incubate at 37°C for 0h 5m 0s
Remove PWB from outer well area and replace with 1mL 30% PWB to outer well area. Incubate at 37°C for 0h 5m 0s
Remove PWB from outer well area and replace with 1mL 30% PWB to outer well area. Incubate at 37°C for 0h 5m 0s
Remove PWB from outer well area and replace with 1mL 2x SSC. Incubate at Room temperature for 0h 5m 0s
Remove 2x SSC from outer well area and replace with 1mL 2x SSC. Incubate at Room temperature for 0h 5m 0s
Prepare Amplifiers
Bring Amplification Buffer (AB) to Room temperature
2µL of a 3 µM stock) of the appropriate amplifier hairpin 1 to 0.2 mL PCR tube. Add 6 pmol (2µL 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 0h 1m 30s .
Remove samples from thermal cycler and place in the dark at Room temperature for 0h 30m 0s
Combine 2µL of hairpin 1 and 2µL of hairpin 2 (and additional hairpins if multiplexing) into 100µL final volume of AB at Room temperature.
Amplification
Remove 2x SSC from the outer and inner well area, then add 100µL of AB to the inner well. Incubate for 0h 5m 0s at Room temperature
Remove AB from the inner well area and replace with 100µL of AB. Incubate for 0h 5m 0s at Room temperature
Remove AB from the inner well area and add 100µL 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 .
Counterstaining, Mounting, and Imaging
Add 2mL 2x SSC to the outer well area (it is not necessary to first remove the amplifier hairpin solution). Incubate 0h 5m 0s at Room temperature
Remove 2x SSC from the outer well area and replace with 2mL 2x SSC. Incubate 0h 5m 0s at Room temperature .
Remove 2x SSC and add 1mL PBS containing 1:100 Hoechst solution. Incubate 0h 20m 0s at Room temperature .
Remove PBS/Hoechst solution and replace with 2mL PBS.
Remove PBS from outer and inner well areas.
Add 100µL Vectashield (or other antifade mounting medium) to the inner well area. Store at 4°C until ready to image.
