Dec 22, 2025
HCR/IF RNA-FISH combined protocol for the whole-mount brains of Drosophila melanogaster
- Amanda A. G. Ferreira1,
- Bogdan Sieriebriennikov2,
- Hunter Whitbeck1,
- Yen-Chung Chen1
- 1New York University;
- 2NYU Grossman School of Medicine
External link: https://doi.org/10.1371/journal.pgen.1010802
Protocol Citation: Amanda A. G. Ferreira, Bogdan Sieriebriennikov, Hunter Whitbeck, Yen-Chung Chen 2025. HCR/IF RNA-FISH combined protocol for the whole-mount brains of Drosophila melanogaster. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld1b4ol5b/v1
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: December 20, 2025
Last Modified: December 22, 2025
Protocol Integer ID: 235559
Keywords: brain, drosophila, ant, hcr, in situ, larva, rna fish, rna fluorescent in situ hybridization, rna fluorescent, hcr rna, drosophila melanogaster, fly drosophila melanogaster, hcr reagent, rna, hcr, other insect, mount samples of the brain, ant harpegnathos saltator, molecular instrument, hybridization chain reaction, jumping ant harpegnathos saltator, different probe set, using hybridization chain reaction, probe, larger concentrations of probe, fish protocol, situ hybridization, mount brains of drosophila melanogaster
Abstract
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).
Citation
LINK
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.
Materials
REAGENTS TO PURCHASE:
Nuclease-Free Water (not DEPC-Treated)Thermo Fisher ScientificCatalog #AM9937
20X PBS (Phosphate Buffered Saline) pH 7.4growcells.comCatalog #MRGF-6396
Schneider's Drosophila MediumThermo FisherCatalog #21720024
Paraformaldehyde, 16% (wt/vol)Electron Microscopy SciencesCatalog #15710
Safety information
Paraformaldehyde is toxic, consult the SDS sheet for proper handling instructions
Note
Avoid long-term storage of the paraformaldehyde solution after opening the ampoule
Triton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100
10% Tween 20Bio-Rad LaboratoriesCatalog #1662404
20X SSCQuality BiologicalCatalog #351-003-131
SlowFade™ Gold Antifade MountantInvitrogen - Thermo FisherCatalog #S36936 - or any other antifade mountant
Hoechst 33258, Pentahydrate (bis-Benzimide), 100 mgThermo FisherCatalog #H1398 - dissolve in DMSO to 5 mg / mL, aliquot and store at -20 ºC
MethanolFisher ScientificCatalog #A412-4
HCR Probe Hybridization BufferMolecular Instruments
HCR Probe Wash BufferMolecular Instruments
HCR Antibody BufferMolecular Instruments HCR 2 Antibody ProbeMolecular Instruments
Safety information
Hybridization and Wash Buffers contain formamide, consult the SDS sheet for proper handling instructions
HCR Amplification BufferMolecular Instruments
Buffer to prepare:
Note
Prepare fresh using nuclease-free water, store at 4 ºC if required after the 1st day of the protocol.
- 1% PBTx (1X PBS with 1% v/v Triton X-100 and 1mM glycine)
- 5X SSCT (5X SSC with 0.1% v/v Tween-20)
- 1X PBS
Troubleshooting
Safety warnings
This protocol uses solutions of paraformaldehyde and formamide, which are highly toxic chemicals. Consult the SDS sheets of the reagents used in this protocol for proper handling instructions.
Day 1
3h 40m
Pre-warm HCR Antibody Buffer to room temperature
300 µL per sample
Dissect brains in cold Schneider's medium
Note
Dissection can also be done in 1X Nuclease-Free PBS.
30m
Fix brains in 500 μL of 4% PFA / Schneider's medium in a spot plate
00:20:00
Room temperature
24 rpm Nutator 60 rpm Orbital Shaker
Note
For fixation and all subsequent steps, samples can be placed either in Eppendorf tubes or in wells of a spot plate (e.g. Pyrex spot plate with 9 depressions, Catalog #CLS722085). Tubes are incubated on a nutator and plates are incubated on an orbital (horizontal) shaker.
20m
Transfer the brains to a PCR tube and rinse 3x with 150 μL of 0.3% PBST
Note
Make 0.3% PBST using 1X Nuclease-Free PBS.
Room temperature
5m
Wash 3x 15 min with 150 μL of 0.3% PBST
Room temperature
00:15:00
00:15:00
00:15:00
24 rpm Nutator
45m
Block samples with 200 μL of HCR Antibody Buffer for 2-4h at 4°C
Note
Blocking time might vary, suggestion for minimum is 1.5h, >2h for optimal signal-to-noise quality
2h
Prepare working concentration of 1° antibodies in HCR Antibody Buffer. Prepare 100 μL per sample
Remove HCR Antibody Buffer and add 100 μL of 1° antibody solution to sample; incubation sample over two nights at 4°C with gentle rotation
4 °C
24 rpm Nutator
Overnight
Note
For fly optical lobe, primary antibody incubation for two nights is optimal; can be adjusted based on the tissue types
Day 3
2h
Pre-warm HCR Antibody Buffer to room temperature
100 µL per sample
Remove excess antibodies by washing 4x30 min with 150 μL of 0.3% PBST at room temperature
Room temperature
00:30:00
00:30:00
00:30:00
00:30:00
24 rpm Nutator
Note
1° Antibody solution can be reused for up to 2-3 times.
2h
Prepare working concentration of HCR Antibody Probe with specific amplifier in 100 μL of HCR Antibody Buffer; antibody probe e.g. anti-mouse B1
Note
Plan out the use of amplifiers for all antibodies and HCR RNA probes for the multiplex experiment; avoid overlap across all antibodies and RNA probes
Remove PBST and add HCR 2° Antibody Probe solution to samples; incubate samples over two nights at 4°C with gentle rotation
4 °C
24 rpm Nutator
Overnight
Day 5
1h 15m
Pre-heat an aliquot of Probe Hybridization Buffer if proceeding with hybridization on the same day (see steps below)
250 µL / sample (If preparing a new probe mixture)
110 µL / sample (If reusing probes)
37 °C
2m
Remove excess 2° Antibody solution by washing 5 x 5 min with 150 μL of 0.3% PBST
00:05:00
00:05:00
00:05:00
00:05:00
00:05:00
Room temperature
24 rpm Nutator
25m
Remove PBST and post-fix sample with 500 μL of 4% PFA, incubate for 10 min at room temperature
Note
Use 500 μL of 4% PFA if transferring back to glass dish, use 200 μL instead if fixing in tube.
10m
Remove fixative and rinse sample quickly 3x with 150 μL of 0.3% PBST
5m
Pre-hybridize samples by incubating them with 100 μL of warm Probe Hybridization Buffer from step 2. Pipet several times to ensure the brains are submerged in Probe Hybridization Buffer instead of residual PBTx.
00:30:00
37 °C
Note
Hybridization buffer is viscous, and the brains might be stuck on the wall or float to the surface. Make sure you see where the brains are during this step. Be careful not to remove them or let them dry out by accident.
30m
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 1µM probe per sample
100 µL Warm probe hybridization buffer per sample
Note
If the signal is weak, consider increase the probe concentration by 2X - 4X.
Remove the pre-hybridization solution from the sample and add the probe solution from step 18.
Note
Take extra care while removing the liquid. Hybridization Buffer is viscous and brains may float.
3m
Incubate samples with the probes.
Overnight We usually do ~24 h, but incubation can be extended to ~48 h. Minimum recommended is 12 h
37 °C
24 rpm Nutator
Day 6
1h 20m
Equilibrate an aliquot of Amplification Buffer to room temperature
Room temperature
60 µL per amplifier per sample (if preparing new) +110 µL per sample
Note
If using 3 different amplifiers for 2 samples, 60 * 3 * 2 + 110 * 2 = 580µL is to be equilibrated.
2m
Pre-heat an aliquot of Probe Wash Buffer
37 °C
450 µL per sample
2m
Remove probe solution and store it at -20°C.
Note
Take extra care while removing the liquid. Hybridization Buffer is viscous and brains may float.
Probe solution can be reused for > 3 times without apparent decrease
Remove excess probes by washing 4 x 10 min with 100µL pre-heated Probe Wash Buffer from step 22
00:10:00 Wash 100µL pre-heated probe wash buffer
00:10:00 Wash 100µL pre-heated probe wash buffer
00:10:00 Wash 100µL pre-heated probe wash buffer
00:10:00 Wash 100µL pre-heated probe wash buffer
37 °C
24 rpm Nutator
40m
Wash samples 2 × 5min with 1mL of 5X SSCT at room temperature.
00:05:00 5x SSCT
00:05:00 5x SSCT
10m
Equlibrate each sample with 100 μL of Amplification Buffer
00:10:00 can be extended to 30 min
Room temperature
24 rpm Nutator or 60 rpm Orbital shaker
10m
During sample equilibration, warm up the thermo cycler to 95°C.
Prepare separately each hairpin (h1 and h2) of each amplifier. Each sample require 3 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.
00:30:00 or longer
Room temperature
Note
For lowly-expressed targets, consider increase the amplifier concentration by 2X.
30m
Briefly spin down the hairpin solutions, add 100 µL/sample amplification buffer, add 2 μL of each hairpin into the 100 µL amplification buffer to make the final mixture.
Room temperature
Remove 100 μL liquid from the samples and add the Amplification Buffer + hairpins from step 29
Note
Take extra care while removing the liquid. Amplification Buffer is viscous and the brain may float.
2m
All the next steps are light sensitive and must be done in the dark!
Incubate in the dark.
Overnight
Room temperature
24 rpm Nutator or 60 rpm Orbital shaker
Day 7
2h 12m
Save hairpin mixture and keep at -20°C
Note
Hairpin mixture can be reused for > 3 times without apparent decrease of signal.
2m
Wash 2 x 5 min and 2 x 15 min with 150 μL of 5X SSCT
00:05:00
00:05:00
00:15:00
00:15:00
Room temperature
24 rpm Nutator
40m
If not using DAPI or GFP/RFP booster, skip this step and the next and proceed to step 37.
If using DAPI: incubate samples with 150 μL of SSCT + DAPI (1:500 from 5 µg/µL stock) at room temperature for 1hr.
If using GFP/RFP booster, add 0.3µL to the butter above (1:500 dilution).
Room temperature
01:00:00
24 rpm Nutator or 60 rpm Orbital shaker
Note
DAPI and GFP/RFP booster incubation works well with 1h RT or 4 degree overnight.
1h
Wash 3 x 10 min with 150 μL of 5X SSCT
00:10:00
00:10:00
00:10:00
Room temperature
24 rpm Nutator
30m
Rinse with 2X Nuclease-Free PBS to remove the detergent; store in PBS with foil to protect sample from light or proceed to mounting and imaging.