Jan 21, 2026

Public workspaceHCR RNA-FISH protocol for the whole-mount antenna of Drosophila

  • Iván Méndez1
  • 1Northwestern University
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Protocol CitationIván Méndez 2026. HCR RNA-FISH protocol for the whole-mount antenna of Drosophila. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3r3pvzp/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: June 29, 2023
Last Modified: January 21, 2026
Protocol Integer ID: 84261
Keywords: drosophila, hcr, in situ, antenna, mount antenna of drosophila, hcr rna, rna fluorescent in situ hybridization, mount samples of drosohpila antenna, drosohpila antenna, rna fluorescent, drosophila, hcr reagent, mount antenna, fish protocol, hcr, rna, hybridization chain reaction, using hybridization chain reaction, molecular instrument, situ hybridization, probe
Abstract
This is a protocol to perform RNA fluorescent in situ hybridization (RNA-FISH) using hybridization chain reaction (HCR) on whole-mount samples of Drosohpila antenna. 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. Modifications include higher amount of probe and hairpins to boost signal, and the description of fixation conditions.

Citation
Meg A. Younger, Margaret Herre, Alison R. Ehrlich, Zhongyan Gong, Zachary N. Gilbert, Saher Rahiel, Benjamin J. Matthews, Leslie B. Vosshall (2020). Non-canonical odor coding ensures unbreakable mosquito attraction to humans. bioRxiv.
LINK

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.
Collection and fixation of antenna
2h 45m
Take aliquots of fixation solution (4%PFA in PBS+3%TritonX) from the -20C and thaw on ice. Put a tube of 1X PBS in ice to cool it down.
Fill a dissection glass with EtOH and a second one with cold PBS.
Collect flies in the fly pad, transfer themto a Falcon tube an put themin ice (this maintains the flies anesthesized but reducing their exposure to CO2).
Before dissecting each fly, dip it quickly in EtOH (this removes the wax covering the cuticle making the fly and antenna to sink in the solutions).
Dissect antenna in cold PBS using forceps. Transfer the antenna using a 20ul pipet with the tip cut, to the fixation solution (keep it in ice). Usually, I dissect as many antenna as I can in 30-40 min - around 24 to 30 antenna is a good number for one in situ run.
After dissecting enough antenna fix for 30 min at RT in fixation solution.
TemperatureRoom temperature Gently shaking Duration00:30:00

Pre-heat an aliquot of Probe Hybridization Buffer @37C if proceeding with hybridization on the same day OR
move to Step 7

30m
Wash antenna before long term storage. To avoid loosing samples during this process, use a dissection scope to remove liquid during washes.

PBT Duration00:05:00
PBT Duration00:05:00
PBT Duration00:05:00
MeOH wash Duration00:05:00
MeOH wash Duration00:05:00
MeOH wash Duration00:05:00

30m
Label with name of species, tissue, and date before storing in MeOH at Temperature-20 °C
In theory, tissues can be stored like this for months, we usually proceed to the HCR within a week

Day 1
1h 45m
For each sample/tube, transfer 550µl of Probe Hybridization Buffer (store at -20C) to a 1.5 mL tube and warm to Temperature37 °C

For all next steps, washes and rinse use around 1ml unless a specific volume is mentioned. Rinse means adding the solution and removing it as soon as the antenna settle in the bottom

Rehydrate samples:
MeOH 75% /PBT Duration00:05:00
MeOH 50% /PBT Duration00:05:00
MeOH 25% /PBT Duration00:05:00
PBT rinse
15m
Post-fix: Incubate for 10 minutes in 4% paraformaldehyde in PBT
Wash:
PBT Duration00:05:00
PBT Duration00:05:00
PBT Duration00:05:00
15m
Pre-hybridize samples in 300 µL of Probe Hybridization Buffer for:
Duration00:30:00 Temperature37 °C
CAUTION: probe hybridization buffer contains formamide, a hazardous material.

30m
In the meantime, prepare a probe solution by adding 8 μL of probe to 200μL of warm Probe Hybridization Buffer. Even when using multiple probes, maintain the final of buffer to 200μL.
Remove the pre-hybridization solution from the sample and add the probe solution from step 9


3m
Add probe mixture and Incubate samples
DurationOvernight around 24h
Temperature37 °C

Overnight
Day 2
1h 20m
Pre-heat an aliquot of Probe Wash Buffer (2mL per tube/sample)
Temperature37 °C

2m
Equilibrate an aliquot of Amplification Buffer (stored at 4C) to room temperature (350ul per sample/tube)
TemperatureRoom temperature

2m
Remove and SAVE PROBE SOLUTION, which can be reused at least 3 times. Save used probe
solutions at Temperature-20 °C
Remove excess probes by washing Temperature37 °C :

400 ul pre-heated Probe Wash Buffer Duration00:10:00
400 ul pre-heated Probe Wash BufferDuration00:10:00
400 ul pre-heated Probe Wash BufferDuration00:10:00

Before the next wash, take hairpins from -20C and thaw on ice

400 ul pre-heated Probe Wash BufferDuration00:10:00
400 ul pre-heated Probe Wash BufferDuration00:10:00

50m
In the meantime, prepare separately each hairpin (h1 and h2) of each amplifier. Add 5 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.
Duration00:30:00 or longer
TemperatureRoom temperature
30m
Critical
Wash samples TemperatureRoom temperature :

500 μL of 5X SSCT Duration00:05:00
500 μL of 5X SSCT Duration00:05:00
500 μL of 5X SSCT Duration00:05:00
15m
Pre-amplify each sample with 250 μL of Amplification Buffer
Duration00:30:00
TemperatureRoom temperature
30m
In the meantime, add 5 μL of each hairpin from step 21 to 100 μL of Amplification Buffer (keep the volume of Amplification Buffer at 100 μL even if multiple hairpin sets are being used)
TemperatureRoom temperature

Remove 250 μL liquid from the samples and add the Amplification Buffer + hairpins from step 18


2m
Incubate in the dark (place tubes inside a cooler and in a drawer)
DurationOvernight
TemperatureRoom temperature
Overnight
Day 3
3h 15m
SAVE HAIRPIN MIXTURE. Can be reused multiple times. Save used hairpin mixtures at -20°C.
20m
During the next washes minimize exposure to light by keeping the tubes in a drawer or inside a cooler.
Wash:

500 μL of 5X SSCT
500 μL of 5X SSCT
500 μL of 5X SSCT
500 μL of 5X SSCT Duration00:15:00
500 μL of 5X SSCT Duration00:05:00
20m
Rinse with 1X Nuclease-Free PBS to remove the detergent
2m
Remove the PBS and add VectaShield.
2m
Mount on slides using circular holders.
10m
Proceed with imaging
Imaging
Citations
Meg A. Younger, Margaret Herre, Alison R. Ehrlich, Zhongyan Gong, Zachary N. Gilbert, Saher Rahiel, Benjamin J. Matthews, Leslie B. Vosshall. Non-canonical odor coding ensures unbreakable mosquito attraction to humans
https://doi.org/10.1101/2020.11.07.368720