TITLE:  Hybridization Chain Reaction combined with Immunohistochemistry for Whole-Mount Embryos
AUTHORS: Ali M Elagoz, Ruth  Styfhals, Sofia Maccuro, Luca Masin, Lieve Moons, Eve  Seuntjens

[DESCRIPTION]
Here, we are providing an optimized, step-by-step clearing protocol that retains the signal generated by HCR v3.0 in whole mount Octopus vulgaris embryos, even in combination with immunohistochemistry. Please cite (Elagoz et al., 2022) if you use this protocol. 

For designing HCR v3.0 probe pairs, we have developed an automated tool called Easy_HCR which can be found at https://github.com/SeuntjensLab/Easy_HCR.



[GUIDELINES]
The bench, lids etc. were cleaned thoroughly with EtOH and RNase away. Work as RNase-free as possible during the duration of the experiment.

[STEPS]
SECTION: Embryo Fixation and Preparation
1. DAY 0

Fix tissue overnight in 4 % paraformaldehyde (PFA) in phosphate-buffered saline (PBS-DEPC) at 4°C.

SECTION: Embryo Fixation and Preparation
2. DAY 1

Wash with PBS-DEPC. 
SECTION: Embryo Fixation and Preparation
3. Dechorionate the octopus embryos in PBST.
SECTION: Dehydration
4. Dehydrate embryos into methanol (MeOH) with a series of graded MeOH/PBST washes for 10 min on ice:
(a) 25% MeOH / 75% PBST
(b) 50% MeOH / 50% PBST
(c) 75% MeOH / 25% PBST
(d) 100% MeOH
(e) 100% MeOH.
SECTION: Dehydration
5. Incubate embryos at -20 °C overnight (> 16 h) or until use.

NOTE: Embryos can be stored for six months at -20 °C.
SECTION: Rehydration and Permeabilization
6. DAY 2

Transfer the required number of embryos for an experiment to a 0.5 ml Eppendorf tube. “Thaw” on ice and gradually move them to room temperature (approx. in half an hour).
SECTION: Rehydration and Permeabilization
7. Rehydrate with a series of graded MeOH/PBST washes for 10 min each on ice:
(a) 75% MeOH / 25% PBST
(b) 50% MeOH / 50% PBST
(c) 25% MeOH / 75% PBST
(d) 100% PBST
(e) 100% PBST
SECTION: Rehydration and Permeabilization
8. Immerse embryos in 10 ug/mL proteinase K solution for 15 min at room temperature (1,08 μl of proteinase K in 2 ml of PBS-DEPC (Proteinase K rec PCR grade, art. 3115887001, Roche)).

NOTE: Proteinase K concentration and treatment time should be reoptimized for each batch of proteinase K, or for samples at a different developmental stage.
SECTION: Rehydration and Permeabilization
9. Wash embryos 2 x 5 min with PBST.
SECTION: Rehydration and Permeabilization
10. 
Postfix with 4% PFA for 20 min at room temperature.

SECTION: Rehydration and Permeabilization
11. 
Wash embryos 3 x 5 min with PBST.
SECTION: Hybridization
12. Pre-warm oven and hybridization buffer to 37°C.
SECTION: Hybridization
13. Remove the buffer and pre-hybridize with 100 ul of probe hybridization buffer for 30 min at 37°C. 
SECTION: Hybridization
14. Thaw probes on ice, spin down before using.
SECTION: Hybridization
15. Prepare probe solution by adding 0.4 pmol of each probe mixture to 100 ul of probe hybridization buffer at 37°C.
SECTION: Hybridization
16. Remove the pre-hybridization solution and add 100 ul of the probe solution and incubate embryos overnight (12–16 h) at 37°C (no shaker is used during this step). 
SECTION: Wash Steps
17. DAY 3

Remove excess probes by washing embryos 4 x 15 min with 100 ul of probe wash buffer at 37°C

NOTE: Pre-warm the wash solution in the oven to 37°C before use.
SECTION: Wash Steps
18. Wash samples 2 x 5 min with 5 x SSCT at room temperature. (Thaw hairpins on ice in the dark and move the amplification buffer to room temp.)
SECTION: Amplification
19. Remove the solution and Pre-amplify embryos with 100 ul of amplification buffer for at least 30 min at room temperature.

NOTE: Equilibrate amplification buffer to room temperature before use.
SECTION: Amplification
20. Separately prepare 6 pmol of H1 and H2 in separate PCR tubes. Specifically, pipet 2 μl of each hairpin [3 μM stock (3 pmol for H1 and 3 pmol for H2) in hairpin storage buffer] in a separate PCR tube. 

SECTION: Amplification
21. In a PCR thermocycler: heat hairpins at 95 °C for 90s. Immediately put hairpins on ice for 5 minutes, and then leave hairpins at room temperature for 30 minutes IN THE DARK.
SECTION: Amplification
22. Prepare the hairpin solution by adding all snap-cooled hairpins to 100 ul of amplification buffer at room T. (Add equal amounts of amplification buffer to both hairpin 1 and 2 and then add hairpin 1 to hairpin 2)
SECTION: Amplification
23. Remove the pre-amplification solution and add the hairpin solution. Incubate the embryos/larvae overnight (12–16 h) in the dark at room temperature (no shaker is used during this step).
SECTION: Wash Steps and Imaging
24. DAY 4

Remove excess hairpins by washing with 100 uL of 5x SSCT at room temperature in the dark:
(a) 2 x 5 min
(b) 2 x 30 min
(c) 1 x 5 min
(d) 1 x 2hrs with DAPI (1:2000)
(e) 1 x 5 mins
SECTION: Wash Steps and Imaging
25. Samples can be transferred to the Fructose-Glycerol clearing solution described in Dekkers et al., 2019 for at least 2 days.

Fructose-Glycerol clearing solution was prepared by dissolving 29,72 grams of fructose in 33 ml of glycerol and 7 ml of distilled water on a magnetic stirrer. The solution can be stored at 4 °C for a month. 
SECTION: Immunohistochemistry (IHC)
26. Incubate embryos with the primary antibody (1:1000 rabbit anti-phosphohistone H3 (Ser10) (Millipore 06-570) for the following 2 days after the HCR protocol. 

IMPORTANT NOTE: When HCR is combined with IHC, the incubation in DAPI is skipped and the embryos are directly processed for IHC after the last excess hairpin removal wash. 

IMPORTANT NOTE: The whole protocol of IHC is carried out at 4°C. 
SECTION: Immunohistochemistry (IHC)
27. DAY 6

Wash embryos with 5xSSCT three times for 2 hours.
SECTION: Immunohistochemistry (IHC)
28. Add the secondary antibody donkey anti-rabbit Alexa 488 (Life Technologies) at a final concentration of 1:300 diluted in antibody diluent (Roche) and incubate O/N. 
SECTION: Immunohistochemistry (IHC)
29. DAY 7

Wash with 5xSSCT twice for 2 hours.
SECTION: Immunohistochemistry (IHC)
30. Incubate embryos in 1:2000 DAPI in 5xSSCT for 2 hours followed by 5xSSCT wash for 5 minutes.
SECTION: Immunohistochemistry (IHC)
31.  Incubate embryos in Fructose-Glycerol clearing as described above before imaging.