TITLE: HCR RNA-FISH protocol for the whole-mount brains of Drosophila melanogaster
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)
250 µL (If preparing a new probe mixture)
100 µL  (If reusing probes)
37 °C 

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

SECTION: Day 1
4. Fix brains in 800 μL of 4% PFA / Schneider's medium
20 min 
Room temperature
  or   
 


SECTION: Day 1
5. Rinse 3x with 150 μL of 1% PBTx
Room temperature 

SECTION: Day 1
6. Wash 3x 15 min with 150 μL of 1% PBTx
15 min
15 min
15 min  
Room temperature 
  or   

SECTION: Day 1
7. Pre-hybridize samples by incubating them with 100 μL of warm Probe Hybridization Buffer from step 2
10 min 
37 °C 

 

SECTION: Day 1
8. In the meantime, prepare a 8nM probe solution by adding 0.4 pmol of each probe mixture (e.g. 0.4 μL of 1 μM stock) to the warm Probe Hybridization Buffer for the total volume of 100 μL.
0.4 µL 
100 µL 


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

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

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

SECTION: Day 2
11. Equilibrate an aliquot of Amplification Buffer to room temperature
Room temperature 
60 µL (if preparing new) +110 µL 


 

SECTION: Day 2
13. Remove excess probes by washing 4 x 10 min with 100µL pre-heated Probe Wash Buffer from step 12
10 min Wash 100µL pre-heated probe wash buffer 
10 min Wash 100µL pre-heated probe wash buffer 
10 min Wash 100µL pre-heated probe wash buffer 
10 min Wash 100µL pre-heated probe wash buffer 
37 °C 
  or   

SECTION: Day 2
17. Prepare separately each hairpin (h1 and h2) of each amplifier. Each sample require 2 μL of each 3 μM stock of hairpin to be mixed in a final volume of 100µL/sample amplification buffer. Aliquot the required amount of hairpin to individual PCR tubes. 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
15. Equlibrate each sample with 100 μL of Amplification Buffer
10 min 
Room temperature  
  or   
SECTION: Day 2
18. Briefly spin down the hairpin solutions, add 100 µL/sample amplification buffer, and use the same buffer to resuspend all hairpins used.
Room temperature

SECTION: Day 2
19. Remove 100 μL liquid from the samples and add the Amplification Buffer + hairpins from step 17
 

SECTION: Day 2
21. Incubate in the dark.
  
37 Room temperature 
  or   
SECTION: Day 3
23. Save hairpin mixture and keep at -20°C
SECTION: Day 3
24. Wash 2 x 5 min with 150 μL of 5X SSCT
5 min 
5 min 
Room temperature  
  or   

SECTION: Day 3
25. Wash 2 x 15 min with 150 μL of 5X SSCT
15 min 
15 min 
Room temperature  
  or   
SECTION: Day 3
26. If not using DAPI, skip this step and the next and proceed to step 28.

If using DAPI: incubate samples with 150 μL of SSCT + DAPI (1:1000 of the 5 mg/mL stock - final concentration 10 μg/mL) for 1 h
120 min 
Room temperature  
  or   
SECTION: Day 3
27. Wash 2 x 10 min with 150 μL of 5X SSCT
10 min
10 min 
Room temperature  
  or   
SECTION: Day 3
28. Rinse with 2X Nuclease-Free PBS to remove the detergent
SECTION: Day 3
29. Remove the PBS and add Slowfade (or another antifade mountant)
SECTION: Day 3
30. 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
31. Proceed with imaging
SECTION: Day 2
14. Wash samples 2 × 5min with 1mL of 5X SSCT at room temperature.
5 min 5x SSCT 
5 min 5x SSCT 

SECTION: Day 2
16. During sample equilibration, warm up the thermo cycler to 95°C.
SECTION: Day 3
22. Prepare 1.2 ml/sample 5X SSCT