TITLE: HCR - Embryo/Larvae fixation and permeabilisation
AUTHORS: FishFloorUCL

[DESCRIPTION]
In situ hybridization chain reaction (HCR) is a method to visualize specific mRNAs in diverse organisms by applying a HCR that is an isothermal enzyme-free nucleotide polymerization method using hairpin DNAs. This specific protocol was developed by Chintan Trivedi based on previous work by Tsuneoka, Y. [1] and Choi [2]. It has many strengths over traditional (F)ISH: probe design is easier and doesn’t require optimisation or purification (contamination is not an issue), it is more specific (no off-target labelling), you can label up to six different targets in the same fish (multiplexing). 

Tsuneoka, Y. and H. Funato, Modified in situ Hybridization Chain Reaction Using Short Hairpin DNAs. Frontiers in Molecular Neuroscience, 2020. 13.
Choi, H.M.T., et al., Third-generationin situhybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development, 2018. 145(12): p. dev165753.

[STEPS]
SECTION: Day 1
7. Wash embryos/larvae 3 x 5 min with 1 mL PBS to stop the fixation
Note: Ensure PFA is disposed of safely 
SECTION: Day 1
8. (Optional) Dissect the brains in PBS
Note: If left undissected, skip to step 8
SECTION: Day 1
9. (Optional - The HCR probes are very small, so HCR will work with no permeabilisation at all, even in undissected brains. Only perform this step if you plan immunostaining or DAPI labelling alongside HCR. Artur does permeabilise for DAPI staining in dissected brains)

Permeabilise, treat with 1 mL of proteinase K (30 µg/mL) for 15 minutes at room temperature.
Note: Optimised for 6 dpf larvae 

Wash with PBST (1 mL each) x2 without incubation (rinse)
SECTION: Day 1
10. (Optional - postfix important for dissected brains to harden and increase structural stability)

Postfix with 1 mL of 4% PFA for 20 minutes at room temperature  
SECTION: Day 1
11. Wash x3 for 5 minutes in 1 mL PBST
SECTION: Day 1 - HCR Detection
12. For each sample, transfer 8 - 10 embryos/larvae to a 1.5 mL Eppendorf tube
Note: Probe concentration optimised for 8 - 10 fish. Less fish is ok, as excess will be washed out 
SECTION: Day 1 - HCR Detection
13. Pre-hybridise with 500 µL of probe hybridization buffer for 30 min at 37° C.
SECTION: Day 1 - HCR Detection
14. Simultaneously, prepare probe solution by adding 2-4 pmol of each probe set (e.g., 2 μL of 1 μM stock) to 500 μL of probe hybridisation buffer at 37° C
Note: For fewer probe pairs (<10), use a higher concentration of probe to improve probe hybridization efficiency
SECTION: Day 0
6. Cull embryos using an overdose of Tricaine (4-5 mL 25X in a 50 mL Petri dish)
Note. Zebrafish larval brain anatomy is standardised at day 6 or 7 post fertilisation.

Fix embryos/larvae in 2 mL of 4% paraformaldehyde (PFA) overnight at 4° C on a rocker (12 – 18 hours)
Note: Use sweet PFA (4% (w/v) sucrose, 1.6b sucrose in 40 mL) for fixing larvae that are to be dissected later
Note: Use fresh PFA (no more than three days old) and cool to 4° C before use to avoid increased autofluorescence
SECTION: Prepare Stock Probe stock solution
5. Plate takes a long time to defrost properly, leave at room temperature for a few hours
Each well in 96 well plate is normalised to 100 µM 

Add 10 uL from each well to 1.5 mL Eppendorf
Make up to 1 mL with nuclease-free water after all the individual probes have been added 
(i.e. 20 probe pairs = 40 individual probes = 40 wells = 400 µL probes + 600 µL water)
15. Remove the pre-hybridisation solution and add the probe solution.
16. Incubate embryos/larvae overnight (12 – 16 hours) at 37° C
Note: Hybridisation timing is flexible – can be left longer if needed
SECTION: Day 2
17. Remove excess probes by washing larvae 4 x 15 min with 500 µL of probe wash buffer at 37°C. 
Note: preheat probe wash buffer to 37° C before use.
SECTION: Day 2
18. Wash embryos/larvae 2 x 5 min with 5x SSCT at room temperature.
SECTION: Day 2 - HCR Amplification
19. Pre-amplify embryos/larvae with 500 µL of amplification buffer for 30 min at room temperature. 
Note: equilibrate amplification buffer to room temperature before use.
SECTION: Day 2 - HCR Amplification
20. Prepare 30 pmol** of each hairpin, h1 and h2, in separate tubes
Snap cool by heating to 95° C for 90 seconds and cool to room temperature for ~30 minutes (there is a protocol in the Themocyclers machines)
**we routinely use 4 µL of each haprin, later diluted in 190 µL amplification buffer, for 6-8 larvae 
HCR hairpins h1 and h2 are provided in hairpin storage buffer ready for snap cooling. 
h1 and h2 should be snap cooled in separate tubes. DO NOT mix them before snap cooling
SECTION: Day 2 - HCR Amplification
21. Prepare the hairpin solution by adding snap-cooled h1 and snap-cooled h2 to 190 µL amplification buffer (for 6-8 larvae)
SECTION: Day 2 - HCR Amplification
22. Remove the pre-amplification buffer solution and add the hairpin solution 
SECTION: Day 2 - HCR Amplification
23. Incubate the embryos/larvae overnight (12-16 hours) in the dark at room temperature.
Note: amplification timing is flexible, can be left longer if needed

SECTION: Day 3
24. Remove excess hairpins by washing with 500 µL SSCT at room temperature:
2 x 5 min
2 x 30 min
1 x 5 min
SECTION: Day 3
25. Wash embryos/larvae with PBS (1 mL each) 2 x without incubation
SECTION: Day 3
26. Samples can be stored in PBS at 4° C protected from light before microscopy for 2-3 days.
SECTION: (Optional) DAPI Staining
27. This protocol is for dissected brains only
Dilute 1 µL DAPI stock (5 mg/mL) in 500 µL 5 x SSCT at room temperature (Final conc 10 µg/mL)
Remove PBS and add DAPI solution
Incubate for 2 hours at room temperature or 4°C overnight 
Wash embryos/larvae with PBS (1 mL each) 2 x without incubation
Samples can be stored at 4° C protected from light before microscopy for 2-3 days
SECTION: Notes
1. Aggressive permeabilization leads to an increase in background autofluorescence
HCR Probes and hairpins do not require excessive permeabilization in dissected brains or whole-mount embryos/larvae 
Transgenic/genetically driven fluorescence (GFP/RFP) persists after the HCR protocol.
SECTION: Buffer Recipes
2. 20X SSC 
Dissolve in 300 mL ddH2O
70.12g NaCl
35.3g Sodium citrate 
Adjust pH to 7.0 with 1M HCl 
Adjust volume to 400 mL
SECTION: Buffer Recipes
3. 5X SSCTw 
12.5 mL 20X SSC 
100 µL 50% Tween Up 
50 mL ddH2O
SECTION: Buffer Recipes
4. 1X PBSTw 
50 mL 1X PBS* 
100 µL 50% Tween   
*no CaCl2 or MgCl2 as it leads to increased autofluorescence