TITLE: HCR RNA-FISH protocol for the whole-mount brains of Drosophila and other insects
AUTHORS: Amanda A. G. Ferreira, Bogdan  Sieriebriennikov, Hunter Whitbeck

[DESCRIPTION]
This is a protocol to perform RNA fluorescent in situ hybridization (RNA-FISH) using hybridization chain reaction (HCR) on whole-mount samples of the brains of the fly Drosophila melanogaster and other insects, e.g. the jumping ant Harpegnathos saltator. Probes and HCR reagents are purchased from Molecular Instruments. This protocol is loosely based on the "generic sample in solution" protocol published by Molecular Instruments. Our modifications include the description of fixation conditions, counterstaining by Hoechst, and altered washes. Additionally, we use larger concentrations of probes and hairpins following the protocol described by Younger, Herre et al. 2020. We have successfully employed this protocol to stain insect brains with up to 4 different probe sets simultaneously (hairpins conjugated with Alexa Fluor 488, 546, 496, and 647).

 


[GUIDELINES]
WORKING PRACTICES:

Prepare all buffers using nuclease-free water. Use filter tips and nuclease-free tubes. If using spot plates, pre-clean them first with household bleach diluted 1:10 in water and then with 70% ethanol. Wear gloves and adhere to other practices aimed at minimizing RNA degradation in the sample. Working in a clean bench is not required if other RNAse-free practice are followed.



PROBE DESIGN:

We select the target sequence or isoform of the gene of interest and let Molecular Instruments design the probes. For genes with multiple isoforms, either target the isoform that includes as many as possible constitutive exons and as few as possible alternatively spliced exons, or the isoform that has the highest RNA-seq coverage (assessed visually in IGV) if RNA-seq data are available. Aim for the highest number of probes in a set, ideally 40, although we have successfully performed experiments with probe sets containing <20 probes.



AMPLIFIER CHOICE:

We routinely perform multiplexed stainings with up to 4 different probe sets and a Hoechst counterstain. We use amplifiers conjugated with Alexa Fluor 488, 546, 594, and 647. We are able to detect clearly distinguishable signals with minimal bleed-through on our confocal microscope (Leica SP8). However, be aware that simultaneously using fluorophores with partially overlapping spectra (e.g. AF 546 and 594) requires setting narrower detection ranges, which reduces the amount of signal detected.

[STEPS]
SECTION: Day 1
1. Prepare all solutions

SECTION: Day 1
2. Pre-heat an aliquot of Probe Hybridization Buffer if proceeding with hybridization on the same day (see step case below)
37 °C 

SECTION: Day 1
3. Dissect brains in cold Schneider's Medium
 

SECTION: Day 1
5. Fix brains in 800 μL of Fixation Buffer
20 min 
Room temperature
  or   
 


SECTION: Day 1
6. Rinse 3x with 500 μL of PBST
Room temperature 

SECTION: Day 1
7. Wash 3x 15 min with 500 μL of PBST
15 min
15 min
15 min  
Room temperature 
  or   

SECTION: Day 1
8. Pre-hybridize samples by incubating them with 250 μL of warm Probe Hybridization Buffer from step 2
10 min 
37 °C 

SECTION: Day 1
9. In the meantime, prepare a 16 nM probe solution by adding 8 pmol of each probe mixture (e.g. 8 μL of 1 μM stock) to the warm Probe Hybridization Buffer for the total volume of 500 μL
 


SECTION: Day 1
10. Remove the pre-hybridization solution from the sample and add the probe solution from step 9
 

SECTION: Day 1
11. Incubate samples with the probes
  
37 °C 
  or   

SECTION: Day 2
12. Pre-heat an aliquot of Probe Wash Buffer 
37 °C 

SECTION: Day 2
13. Equilibrate an aliquot of Amplification Buffer to room temperature
Room temperature 

SECTION: Day 2
14. Remove excess probes by washing 5x 10 min with pre-heated Probe Wash Buffer from step 12
10 min 
10 min 
10 min 
10 min 
10 min 
37 °C 
  or   

SECTION: Day 2
15. In the meantime, prepare separately each hairpin (h1 and h2) of each amplifier. Add 7 μL / sample of the 3 μM stock of each hairpin to a separate PCR tube. For example, if amplifiers B1 and B2 are being used, prepare 4 PCR tubes that contain B1-h1, B1-h2, B2-h1, and B2-h2, respectively. Incubate the tubes in a thermocycler at 95 ºC for 90 sec. Immediately take them out of the machine (while it is still at 95 ºC), place them in a rack and incubate them at room temperature in the dark for at least 30 min.
30 min 
Room temperature  
SECTION: Day 2
16. Wash samples 2x 5 min with 500 μL of 5X SSCT
5 min  
5 min  
Room temperature  
  or   
SECTION: Day 2
17. Pre-amplify each sample with 250 μL of Amplification Buffer
10 min 
Room temperature  
  or   
SECTION: Day 2
18. In the meantime, add 6 μL of each hairpin from step 15 to 100 μL of Amplification Buffer (keep the volume of Amplification Buffer at 100 μL even if multiple hairpin sets are being used) 
Room temperature

SECTION: Day 2
19. Remove 250 μL liquid from the samples and add the Amplification Buffer + hairpins from step 18
 

SECTION: Day 2
21. Incubate in the dark  
  
37 Room temperature 
  or   
SECTION: Day 3
22. Equilibrate an aliquot of Probe Wash Buffer to room temperature
Room temperature 

SECTION: Day 3
23. Add 300 μL of 5X SSCT to samples, which at this point still contain Amplification Buffer + hairpins. This will make the solution inside the tubes less viscous. Remove as much liquid as possible.
SECTION: Day 3
24. Wash 1x 5 min with 500 μL of 5X SSCT
5 min 
Room temperature  
  or   

SECTION: Day 3
25. Wash 1x 15 min with 500 μL of Probe Wash Buffer
15 min 
Room temperature  
  or   
SECTION: Day 3
26. If not using Hoechst, skip this step and the next and proceed to step 28.

If using Hoechst: incubate samples with 500 μL of Probe Wash Buffer + Hoechst (1 μL of the 5 mg/mL stock - final concentration 10 μg/mL) for 2 h
120 min 
Room temperature  
  or   
SECTION: Day 3
27. Wash 1x 15 min with 500 μL of Probe Wash Buffer
15 min 
Room temperature  
  or   
SECTION: Day 3
28. Wash 1x 10 min with 500 μL of 5X SSCT
10 min 
Room temperature  
  or   
SECTION: Day 3
29. Rinse with 1X Nuclease-Free PBS to remove the detergent
SECTION: Day 3
30. Remove the PBS and add Slowfade (or another antifade mountant)
SECTION: Day 3
31. If necessary, finish dissecting to remove extra tissue. Mount brains on slides.
SECTION: Day 2
20. All the next steps are light sensitive and must be done in the dark!
SECTION: Day 3
32. Proceed with imaging
SECTION: Day 1
None. 
SECTION: Day 1
4. Choose the version of the protocol you would like to follow (Fly is default)
SECTION: Day 1
5. Fix brains in 500 μL of Fixation Buffer
20 min 
4 °C
  or   
 


SECTION: Day 2
6. Wash 3x 10 min with 500 μL of PBST
10 min
10 min
10 min  
4 °C 
  or   

SECTION: Day 3
13. Pre-hybridize samples by incubating them with 500 μL of warm Probe Hybridization Buffer from step 12
30 min 
37 °C 

SECTION: Day 3
14. In the meantime, prepare a 16 nM probe solution by adding 8 pmol of each probe mixture (e.g. 8 μL of 1 μM stock) to the warm Probe Hybridization Buffer for the total volume of 500 μL



SECTION: Day 3
15. Remove the pre-hybridization solution from the sample and add the probe solution from step 14
 

SECTION: Day 3
16. Incubate samples with the probes
  
37 °C 
  or   

SECTION: Day 4
18. Equilibrate an aliquot of Amplification Buffer to room temperature
Room temperature 

SECTION: Day 4
19. Remove excess probes by washing 5x 10 min with pre-heated Probe Wash Buffer from step 17
10 min 
10 min 
10 min 
10 min 
10 min 
37 °C 
  or   

SECTION: Day 4
20. In the meantime, prepare separately each hairpin (h1 and h2) of each amplifier. Add 7 μL / sample of the 3 μM stock of each hairpin to a separate PCR tube. For example, if amplifiers B1 and B2 are being used, prepare 4 PCR tubes that contain B1-h1, B1-h2, B2-h1, and B2-h2, respectively. Incubate the tubes in a thermocycler at 95 ºC for 90 sec. Immediately take them out of the machine (while it is still at 95 ºC), place them in a rack and incubate them at room temperature in the dark for at least 30 min.
30 min 
Room temperature  
SECTION: Day 4
21. Wash samples 2x 5 min with 500 μL of 5X SSCT
5 min  
5 min  
Room temperature  
  or   
SECTION: Day 4
22. Pre-amplify each sample with 500 μL of Amplification Buffer
30 min 
Room temperature  
  or   
SECTION: Day 4
23. In the meantime, add 6 μL of each hairpin to 100 μL of Amplification Buffer (keep the volume of Amplification Buffer at 100 μL even if multiple hairpin sets are being used) 
Room temperature

SECTION: Day 4
24. Remove liquid from the samples and add the Amplification Buffer + hairpins from step 23
 

SECTION: Day 4
25. All the next steps are light sensitive and must be done in the dark!
SECTION: Day 4
26. Incubate in the dark  
  
37 Room temperature 
  or   
SECTION: Day 5
27. Add 300 μL of 5X SSCT to samples, which at this point still contain Amplification Buffer + hairpins. This will make the solution inside the tubes less viscous. Remove as much liquid as possible.
SECTION: Day 5
28. Incubate samples with 500 μL of Probe Wash Buffer + Hoechst (1 μL of the 5 mg/mL stock - final concentration 10 μg/mL) for 1.5 h
90 min 
Room temperature  
  or   
SECTION: Day 5
30. Wash 2x 5 min with 500 μL of 5X SSCT
5 min 
5 min 
Room temperature  
  or   
SECTION: Day 5
31. Mount brains on slides in Slowfade (or another antifade mountant)
SECTION: Day 5
32. Proceed with imaging
SECTION: Day 2
7. Dehydrate in a graded series of MeOH/PBST washes (add 500 μL at each step):
25% MeOH : 75% PBST 10 min 
50% MeOH : 50% PBST 10 min 
75% MeOH : 25% PBST 10 min 
100% MeOH : 0% PBST 10 min 
4 °C 

SECTION: Day 2
8. Replace 100% MeOH in the tube and incubate at -20 ºC
  
-20 °C 

SECTION: Day 2
9. If desired, samples can be stored at -20 ºC long-term (for months and possibly longer).
SECTION: Day 3
10. Rehydrate in a graded series of MeOH/PBST washes (add 500 μL at each step):
75% MeOH : 25% PBST 10 min 
50% MeOH : 50% PBST 10 min 
25% MeOH : 75% PBST 10 min 
0% MeOH : 100% PBST 10 min 
4 °C 

SECTION: Day 3
11. Wash 1x 10' with 500 μL of PBST
10 min 
Room temperature 

SECTION: Day 3
12. In the meantime, pre-heat an aliquot of Probe Hybridization Buffer
37 °C 

SECTION: Day 4
17. Pre-heat an aliquot of Probe Wash Buffer 
37 °C 
SECTION: Day 5
29. Repeat step 28:
Add 500 μL of Probe Wash Buffer + Hoechst (1 μL of the 5 mg/mL stock - final concentration 10 μg/mL) and incubate for 1.5 h
90 min 
Room temperature  
  or