All methods derived originally from Albertin Lab D. pealeii HCR protocol (unpublished).

Table of Contents

• Equipment
• Reagents
• Buffer Recipes
• Protocol
  • Day 1
  • Day 2
  • Day 3

Equipment

• Water bath (or hyb oven)
• Heat block

Reagents

Homemade

• 4% PFA in sea water
• 1% PBS-T
• High-SDS Hybridization Buffer (High-SDS HYB)
• 5x SSCT

Molecular Instruments provided

• Probe Wash Buffer
• Hybridization Buffer
• Amplification Buffer

Buffer Recipes

High-SDS HYB (for 500mL)

5x SSCT (for 200mL)

Protocol

Embryo collection and fixation

1. Fix embryos overnight in 4% PFA in sea water at 4°C in glass vial.

• Can be left for up to 4 days

2. Warm up High-SDS HYB to 37ºC to resuspend SDS.
3. Wash embryos in PBS-T

• 2x 5 min
• 3x 30 min

4. Remove PBS-T and add High-SDS HYB (~5x volume of embryos).
5. Wait for embryos to settle to the bottom of the vial.
6. PAUSE POINT: Store at -20ºC

Day 1

Preparation

1. Warm water bath (or hyb oven) to 37ºC.
2. Warm MI HYB to 37ºC.
3. Thaw vials with fixed embryos at room temperature.

Prepare Probe Solution

• The probe solution (prepared in MI HYB) should be a total of 150µl per hyb/eppendorf tube.
• If you have multiple samples with the same probe combination, you can prepare a mastermix

1. Prepare as follows
   • For each probe: 3µL of each probeset (from 1µM working stock) * $n_{samples}$
   • For MI HYB: (150µL - (3µL # probesets)) $n_{samples}$
   • Final probe concentration should be 20nM
2. Vortex and spin down to collect
3. Incubate probe solution at 37ºC until embryos are ready.

Adding probe solution to embryos

1. Transfer 2-3 embryos into a 2ml Eppendorf tube.
2. Remove remaining High-SDS HYB.
3. Add 200µL of MI HYB
4. Incubate for ≥30 min at 37ºC.
5. Remove MI HYB solution and add 150µL probe solution.
6. Incubate overnight at 37ºC.

Day 2

Preparation

1. Warm probe wash buffer to 37ºC.
2. Warm Amplification Buffer to room temperature (Not 37ºC)

Washes

1. Remove probe solution from embryos
   • Save probes in a labeled tube and store at -20ºC for reuse
2. Wash embryos in 400µL warm probe wash buffer at 37ºC.
   • 2x 5 min
   • 2x 15 min
   • 2x 30 min
3. Wash embryos in 1mL warm probe wash buffer at 37ºC for 1h
   • In the meantime, prepare the hairpins
4. Remove samples from water bath/heat block
   • Set hyb oven temp to 25ºC
5. Wash embryos in 500µL 5x SSCT at room temp.
   • 2x 5 min
6. Incubate embryos in 500µL Amplification Buffer at room temperature for ≥20min.

Prepare Hairpins

1. Set heat block to 95ºC.
2. Prepare hairpin mix:
   • Place 3µL * $n_{samples}$ of each hairpin (make sure to have h1 and h2 for each channel) in a tube
3. Heat shock hairpins at 95ºC for 90 sec.
4. Place hairpins in a dark drawer for ≥30 min to anneal
5. Add 150µL * $n_{samples}$ of Amplification Buffer (room temp) to the hairpin mix.
6. Vortex briefly and spin down
7. Leave in the dark until needed.

Incubate embryos with hairpins

1. Remove Amplification Buffer from embryos
2. Add 150µL of hairpin solution
3. Incubate overnight at 25ºC.

Day 3

1. Remove hairpins and store in a labeled tube at -20ºC (hairpins can be reused)
2. Wash embryos in 600µL 5x SSCT on rocker at room temp.
   • 2x 5 min
   • 2x 15 min
   • 2x 30 min (optional: add 1:1000 DAPI in the first 30 min wash)
3. Store embryos in 5x SSCT (NOT PBS) at 4ºC until imaging.

References

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