Jan 21, 2026
HCR RNA-FISH protocol for the whole-mount antenna of Drosophila
- Iván Méndez1
- 1Northwestern University
External link: https://doi.org/10.1371/journal.pgen.1010802
Protocol Citation: Iván 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
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.
Room temperature Gently shaking 00:30:00
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 00:05:00
PBT 00:05:00
PBT 00:05:00
MeOH wash 00:05:00
MeOH wash 00:05:00
MeOH wash 00:05:00
30m
Label with name of species, tissue, and date before storing in MeOH at -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 37 °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 00:05:00
MeOH 50% /PBT 00:05:00
MeOH 25% /PBT 00:05:00
PBT rinse
15m
Post-fix: Incubate for 10 minutes in 4% paraformaldehyde in PBT
Wash:
PBT 00:05:00
PBT 00:05:00
PBT 00:05:00
15m
Pre-hybridize samples in 300 µL of Probe Hybridization Buffer for:
00:30:00 37 °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
Overnight around 24h
37 °C
Day 2
1h 20m
Pre-heat an aliquot of Probe Wash Buffer (2mL per tube/sample)
37 °C
2m
Equilibrate an aliquot of Amplification Buffer (stored at 4C) to room temperature (350ul per sample/tube)
Room temperature
2m
Remove and SAVE PROBE SOLUTION, which can be reused at least 3 times. Save used probe
solutions at -20 °C
Remove excess probes by washing 37 °C :
400 ul pre-heated Probe Wash Buffer 00:10:00
400 ul pre-heated Probe Wash Buffer00:10:00
400 ul pre-heated Probe Wash Buffer00:10:00
Before the next wash, take hairpins from -20C and thaw on ice
400 ul pre-heated Probe Wash Buffer00:10:00
400 ul pre-heated Probe Wash Buffer00:10:00
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.
00:30:00 or longer
Room temperature
30m
Wash samples Room temperature :
500 μL of 5X SSCT 00:05:00
500 μL of 5X SSCT 00:05:00
500 μL of 5X SSCT 00:05:00
15m
Pre-amplify each sample with 250 μL of Amplification Buffer
00:30:00
Room 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)
Room 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)
Overnight
Room temperature
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 00:15:00
500 μL of 5X SSCT 00:05:00
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
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