HCR RNA in situ hybridization with whole mount Drosophila brains

This work is licensed under CC BY-SA 4.0

Process

Avoid RNase contamination throughout the process. For example, wear gloves and lab coat all the time, use filtered RNase-free pipette tips, and keep bench surfaces clean.

Cover the samples at all times to prevent fluorophore bleaching.

Do experiment and control group samples in the same batch. Prepare master mixes wherever possible.

This protocol uses commericial HCR probe and amplifier oligos from Molecular Instruments, Inc.

If you find this protocol useful, please cite: https://doi.org/10.3389/fphys.2022.1051544

Day 1: dissection and permeablization

total/hands-on time ~ 1.5hr/1hr

1. Prepare 4% paraformaldehyde (PFA) in 1xPBS for fixation by diluting the 16% PFA stock with 1xPBS. Each sample requires 500uL.
2. Prepare 70% ethanol in ultrapure water for permeablization. Each sample requires 1mL.
3. Clean glass spot plates with distilled water and 70% ethanol. Wipe the plates dry with paper towel.
4. Pipette 400uL of 4% PFA into one spot of the dish. Mark the dish and put on ice.
5. Dissect 10 brains within 30min. Transfer dissected brains into the dish on ice.
6. Nutate for 20min at room temperature for fixation. Fixation time shall be accurate to prevent overfixing.
7. Rinse the sample three times with RNase-free 1xPBS. Do not touch the brains with pipette tips to prevent sample loss.
8. Wash the sample two times with RNase-free 1xPBS, 5min on nutator for each wash.
9. Carefully remove the wash as much as possible.
10. Immediately add 400uL of ice-cold 70% ethanol and transfer the brains into a 1.5mL tube. Fill with 70% ethanol to a final volume of 1mL. Keep the tube on ice throughout.
11. Incubate at 4C overnight to permeablize the sample. While the samples are stable for at least two days in 4C, it is advisable to continue on the next day.

Day 2: hybridize HCR probe

total/hands-on time ~ 0.5hr

1. Prepare RNA-FISH wash buffer (see Material). Each sample requires 1mL.
2. Prepare hybridization buffer without probe (see Material). Each sample requires 200uL.
3. Carefully remove 70% ethanol and add 1mL of wash buffer.
4. Incubate at room temperature for 5min.
5. Carefully remove the wash buffer and add 100uL hybridization buffer without probe.
6. Incubate at 37C for 30min to equilibrate¹.
7. Thaw probe and mix well. Add probes to the spare hybridization buffer (100uL/sample). Vortex briefly and invert to mix. Leave at 37C to allow buffer to warm up.
8. After 30min equilibration, exchange into hybridization buffer with probe.
9. Incubate at 37C overnight (>16hr) to allow sufficient hybridization.

Day 3: amplification with hairpin

total/hands-on time ~ 2hr/0.5hr

1. Prepare RNA-FISH wash buffer. Each sample requires 2mL. Warm the wash buffer to 37C.
2. Prepare amplification buffer (see Material). Each sample requires 200uL.
3. Carefully remove the hybridization buffer and add 1mL of wash buffer. The brains will be semi-transparent so use caution.
4. Incubate at 37C for 30min. Keep the remaining wash buffer at 37C.
5. Carefully repeat the wash once more with 1mL of wash buffer.
6. Carefully remove the wash buffer as much as possible. Aim to leave no more than 10uL so use P200 as needed.
7. Add 100uL of amplification buffer without amplifier hairpins. Incubate at room temperature for 30min to equilibrate.
8. Thaw amplifier hairpins at room temperature. Each probe shall match to one pair of amplifier hairpins. Vortex, spin down, and keep on ice. Cover to protect from light.
9. Pipette 2uL of each hairpin into a 0.2mL PCR tube for each sample.
10. Heat amplifier hairpins at 95C for 1.5min and snap on ice². Each hairpin must be heated separately. Use PCR cycler with heated lid on to prevent evaporation.
11. Add amplifiers to the spare amplification buffer (100uL/sample). Vortex briefly and invert to mix. Store at RT and protect from light.
12. After 30min equilibration, exchange into amplification buffer with amplifiers.
13. Nutate samples overnight at room temperature to allow amplification. Protect from light. Final signal strength will depend on incubation time before saturation.

Day 4: mounting

total/hands-on time ~ 1.5hr/0.5hr

1. Carefully remove amplification buffer.
2. Wash two times each with 1mL of 2xSSCTw (see Material), 30min room temperature each. Do not add RVC³.
3. Remove 2xSSCTw and add 200uL of 2xSSC. The sample is now ready for mounting.
4. It is advisable to use a glycerol-based mountant such as VectaShield or Prolong. After mounting leaving the slide at room temperature overnight will partially clear the tissue and yield better signal.

Material and Storage

Working solution

When preparing working solution, use 10% Tween-20 and 20% dextran sulfate stock solution.

Warm 200mM RVC stock aliquots at 37C until they completely dissolve (will be transparent).

wash buffer

2xSSC, 10% formamide, 2mM RVC, 0.25% Tween-20

hybridization buffer

2xSSC, 10% formamide, 10% dextran sulfate, 2mM RVC, 0.25% Tween-20, 5nM probe (add 0.5uL 1uM probe stock per 100uL final buffer)

amplification buffer

2xSSC, 10% dextran sulfate, 2mM RVC, 0.25% Tween-20, 60nM each amplifier (add 2uL 3uM amplifier stock per 100uL final buffer)

2xSSCTw

2xSSC, 0.25% Tween-20

A quick note on preparing 2xSSCTw: to prepare 40mL, mix 35mL water, 4mL 20xSSC, 1mL 10% Tween-20.

Stock reagent

For Tween-20, prepare 10% (v/v) stock using ultrapure water which will aid pipetting.

For dextran sulfate, prepare 20% (w/v) stock using ultrapure water. Overlay dextran sulfate powder between water layers and put in 4C overnight to allow dissolving.