TITLE: HCR-fluorescent in situ hybridization (HCR-FISH) of gemmule-hatched freshwater sponges
AUTHORS: Scott Nichols

[DESCRIPTION]
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

[GUIDELINES]
We take reasonable measures to use RNAse-free reagents, but have successfully used this protocol with and without filter-tips.

[STEPS]
SECTION: Grow sponges in coverslip bottom dishes
1. 
 
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. 

SECTION: Membrane counterstaining and fixation
3. Remove culture medium from the outer well and inner well areas by aspiration or pipetting. 
 


SECTION: Treat with RNAse inhibitor
8. 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)
SECTION: Dehydrate
15. 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 5 min at Room temperature


 

SECTION: Rehydrate and Permeabilize
19. 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 5 min at Room temperature
 

SECTION: Proteinase K treatment
24. Remove the PTw from the outer well area and replace with 1 mL  of 5 µg/mL Proteinase K (in PTw) for 90 s at Room temperature 

SECTION: Post-fixation and Wash
29. Remove PTw from outer well area and replace with 3 mL of 4% paraformaldhyde in PTw. Incubate for 60 min at Room temperature .

 


SECTION: Prehybridization
34. Bring 30% hybridization buffer (HB) to Room temperature 

 

SECTION: Probe hybridization
38. During Prehybridization, prepare probe solutions.
 

SECTION: Washes
41. Heat 30% Probe Wash Buffer (PWB) to 37 °C (4 mL per sample).

SECTION: Prepare Amplifiers
48. Bring Amplification Buffer (AB) to Room temperature 



SECTION: Amplification
54. Remove 2x SSC from the outer and inner well area, then add 100 µL of AB to the inner well. Incubate for 5 min at Room temperature 

SECTION: Counterstaining, Mounting, and Imaging
59. Add 2 mL 2x SSC to the outer well area (it is not necessary to first remove the amplifier hairpin solution). Incubate 5 min  at Room temperature 

SECTION: Grow sponges in coverslip bottom dishes
2. Grow the sponges for 10080 min in the dark (this reduces the growth of Chlorella-like algal symbionts that autofluoresce (particularly in the far-red channel). 
 


SECTION: Membrane counterstaining and fixation
5. To wash, it is not necessary to remove the staining solution. Instead, add 3 mL lake/spring water directly to the outer well area.

SECTION: Membrane counterstaining and fixation
4. Add 90-100 µL of 1:1000 Cellbrite-Fix 640 (Biotius) in lake/spring water to the inner well area for 15 min at Room temperature in the dark. 
 

 


SECTION: Membrane counterstaining and fixation
6. Immediately remove the wash by pipetting or aspiration from the outer well area.

SECTION: Membrane counterstaining and fixation
7. Add 3 mL of cold 4% paraformaldehyde in 1/4x HF Buffer to the outer well area. Place at 4 °C   in the dark.

 

 



SECTION: Treat with RNAse inhibitor
9. Add 4 mLs of 1/4x HF Buffer to the outer well area of the dish. Incubate 5 min 

SECTION: Treat with RNAse inhibitor
10. Remove the wash from the outer well area, and repeat step 9.
SECTION: Treat with RNAse inhibitor
11. Remove the wash from the outer and inner well areas (be very careful pipetting near the tissue). 
SECTION: Treat with RNAse inhibitor
12. 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 10 min .

SECTION: Treat with RNAse inhibitor
13. Wash by adding 3 mL ¼ x HF Buffer to the outer well area for 3 min at Room temperature . 

SECTION: Treat with RNAse inhibitor
14. Remove wash by pipetting from the outer well area only, then repeat wash (step 13) once.
SECTION: Dehydrate
16. Remove 30% Methanol solution from the outer well area and add 2 mL of 70% Methanol diluted in ¼ x HF Buffer. Incubate 5 min at Room temperature


SECTION: Dehydrate
17. Remove 70% Methanol solution from the outer well area and add 3 mL of 100% Methanol. Incubate 5 min at Room temperature


 


SECTION: Dehydrate
18. Remove the Methanol from the outer well area and replace with 3 mL  100% Ethanol. Incubate 5 min at Room temperature

SECTION: Rehydrate and Permeabilize
20. Remove the 70% Ethanol from the outer well area and replace with 2 mL  of 30% Ethanol diluted in PTw. Incubate 5 min at Room temperature
 
SECTION: Rehydrate and Permeabilize
21. Remove the 30% Ethanol from the outer well area and replace with  2 mL  of 30% Ethanol diluted in PTw. Incubate 5 min at Room temperature
 
SECTION: Rehydrate and Permeabilize
22. Remove 30% Ethanol from the outer well area and replace with 2 mL of PTw. Incubate 5 min at Room temperature 

SECTION: Proteinase K treatment
25. 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 5 min at  5 Room temperature 

SECTION: Proteinase K treatment
26. Remove the glycine solution from the outer well area and add an additional 2 mL  of 2 mg/mL glycine (in PTw) for  5 min at  5 Room temperature 
SECTION: Proteinase K treatment
27. Remove the glycine solution from the outer well area and replace with  2 mL  of PTw for  5 min at  5 Room temperature 
SECTION: Proteinase K treatment
28. Remove the PTw from the outer well area and add an additional  2 mL  of PTw for  5 min at  5 Room temperature 
SECTION: Rehydrate and Permeabilize
23. Repeat PTw wash, 1 time.
SECTION: Post-fixation and Wash
30. Remove fixative from the outer well area, and replace with 3 mL of PTw. Incubate for 10 min at Room temperature .

SECTION: Post-fixation and Wash
31. Remove PTw from outer well area and add an addition 3 mL PTw to the outer well area. Incubate for 10 min at Room temperature .

SECTION: Post-fixation and Wash
32. Remove PTw from outer well area and replace with 3 mL of 2x SSC. Incubate for 10 min at Room temperature 

SECTION: Post-fixation and Wash
33. Remove 2x SSC from the outer well area and replace with 3 mL of 2x SSC. Incubate for 10 min at Room temperature 
SECTION: Prehybridization
35. Remove 2x SSC from outer and inner well area.
SECTION: Prehybridization
36. Add 1 mL HB to the dish. Incubate for 10 min at Room temperature 

SECTION: Prehybridization
37. Remove HB from outer well area and replace with 1 mL HB. Incubate for 30 min at 37 °C 

SECTION: Probe hybridization
39. 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.

SECTION: Probe hybridization
40. Incubate on a very gently rocking platform at 37 °C   .

 


SECTION: Washes
42. 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 5 min 

SECTION: Washes
43. Remove PWB from outer well area and replace with 1 mL  30% PWB to outer well area. Incubate at 37 °C for 5 min 
SECTION: Washes
44. Remove PWB from outer well area and replace with  1 mL  30% PWB to outer well area. Incubate at 37 °C for 5 min 
SECTION: Washes
45. Remove PWB from outer well area and replace with 1 mL  30% PWB to outer well area. Incubate at 37 °C for 5 min 
SECTION: Washes
46. Remove PWB from outer well area and replace with 1 mL 2x SSC. Incubate at Room temperature for 5 min 

SECTION: Washes
47. Remove 2x SSC from outer well area and replace with  1 mL 2x SSC. Incubate at Room temperature for 5 min 
SECTION: Prepare Amplifiers
49. 
 
Add 6 pmol (2 µL of a 3 µM stock) of the appropriate amplifier hairpin 1 to 0.2 mL PCR tube.


SECTION: Prepare Amplifiers
50. Add 6 pmol (2 µL of a 3 µM stock) of the appropriate amplifier hairpin 2 to a 0.2 mL PCR tube.
SECTION: Prepare Amplifiers
51. Add amplifier samples to preheated 95 °C thermal cycler for exactly 90 s .

SECTION: Prepare Amplifiers
52. Remove samples from thermal cycler and place in the dark at Room temperature for 30 min 

SECTION: Prepare Amplifiers
53. 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.

 


SECTION: Amplification
55. Remove AB from the inner well area and replace with 100 µL of AB. Incubate for 5 min at Room temperature 

SECTION: Amplification
56. Remove AB from the inner well area and add 100 µL of the hairpin solution from step 53.
SECTION: Amplification
57. 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.
SECTION: Amplification
58. Incubate in the dark at Room temperature    .

SECTION: Counterstaining, Mounting, and Imaging
60. Remove 2x SSC from the outer well area and replace with 2 mL 2x SSC. Incubate 5 min  at Room temperature . 

SECTION: Counterstaining, Mounting, and Imaging
61. Remove 2x SSC and add 1 mL PBS containing 1:100 Hoechst solution. Incubate 20 min at Room temperature .

SECTION: Counterstaining, Mounting, and Imaging
62. Remove PBS/Hoechst solution and replace with 2 mL PBS.

SECTION: Counterstaining, Mounting, and Imaging
63. Remove PBS from outer and inner well areas.
SECTION: Counterstaining, Mounting, and Imaging
64. Add 100 µL Vectashield (or other antifade mounting medium) to the inner well area. Store at 4 °C until ready to image.