TITLE: HCR in the larval zebrafish eye
AUTHORS: Stephen Carter

[DESCRIPTION]
This protocol describes hybridisation chain reaction (HCR) in situs in the eyes of zebrafish larvae >4 dpf. It is broadly the same as the standard protocol from molecular instruments, with modifications to improve staining in the eye. This is necessary due to the poorer penetration of reagents into the eye compared to other tissues, such as the brain. 

[STEPS]
SECTION: Sample Preparation
1. Raise larvae in embryo medium containing 0.003% of 1-phenyl 2-thiourea (PTU) to inhibit pigment development. Alternatively, bleaching with H2O2 can be performed post fixation, however our experience has so far been that better quality images are obtained with PTU-treated fish. 

PTU cannot completely inhibit RPE pigmentation at non-teratogenic concentrations, however the residual pigment still present tends to be cleared by the HCR buffers. 
SECTION: Sample Preparation
2. Fix larvae in 4% paraformaldehyde (PFA) for 1-2 hours at room temperature.
 

SECTION: Sample Preparation
3. Wash 3 x 5 min with 1X PBS
SECTION: Sample Preparation
5. Permeabilise larvae by incubation in 100% methanol for 30 min at room temperature
 

SECTION: Sample Preparation
6. Rehydrate 

1 wash in 50% methanol/PBST x 5 min

3 x 5 min washes in 1X PBST
SECTION: Sample Preparation
7. Optional:

If greater permeabilisation is required, proteinase K or 80% acetone treatement may be performed here. 
SECTION: Hybridisation
8. Pre-hybridise larvae by incubation for 30 minutes in 500 µL of hybridisation buffer at 37 °C. 
SECTION: Hybridisation
9. Prepare probe solution by adding 10 pmol of each probe set (e.g. 10 µL of  1 micromolar (µM)stock) to 500 µL of probe hybridization buffer at 37 °C. 
SECTION: Hybridisation
10. Remove the pre-hybridization solution and add the probe solution. Incubate larvae for 2 days at 37 °C.
 

SECTION: Hybridisation
11. Remove excess probes by washing embryos/larvae 4 x 15 min with 500 µL of probe wash buffer at 37 °C.
SECTION: Hybridisation
12. Wash embryos/larvae 2 x 5 min with 5X SSCT at room temperature.
SECTION: Amplification
13. Pre-amplify larvae with 500 µL of amplification buffer for 30 min at room temperature.
SECTION: Sample Preparation
4. Optional:

Bleaching may be performed here.
Incubate larvae in a solution of 3% H2O2, 0.5% KOH in water. Monitor until most pigment has been removed and then stop bleaching by washing with PBS until no more bubbles appear in the solution. 
SECTION: Amplification
14. Separately prepare 15 pmol of hairpin h1 and 15 pmol of hairpin h2 by snap cooling 5 µL of3 micromolar (µM) stock (heat at 95 °C for 90 seconds and cool to room temperature in a dark drawer for 30 min).
SECTION: Amplification
15. Prepare hairpin solution by adding snap-cooled h1 hairpins and snap-cooled h2 hairpins to 250 µL of amplification buffer at room temperature.
SECTION: Amplification
16. Remove the pre-amplification solution and add the hairpin solution. Incubate the larvae for 2-3 days in the dark at room temperature. 2 days is usually sufficient for good staining. 
SECTION: Amplification
17. Remove excess hairpins by washing with 500 µL of 5X SSCT at room temperature:
(a) 2 x 5 min
(b) 2 x 30 min
(c) 1 x 5 min
SECTION: Amplification
18. Samples can be stored in 5X SSCT for at day or two at 4 °C.