Hybridization Chain Reaction combined with Immunohistochemistry for Whole-Mount Embryos

DAY 0

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

DAY 1

Wash with PBS-DEPC.

Dechorionate the octopus embryos in PBST.

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.

Incubate embryos at -20 °C overnight (> 16 h) or until use.

NOTE: Embryos can be stored for six months at -20 °C.

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).

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

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.

Wash embryos 2 x 5 min with PBST.

Postfix with 4% PFA for 20 min at room temperature.

Wash embryos 3 x 5 min with PBST.

Pre-warm oven and hybridization buffer to 37°C.

Remove the buffer and pre-hybridize with 100 ul of probe hybridization buffer for 30 min at 37°C.

Thaw probes on ice, spin down before using.

Prepare probe solution by adding 0.4 pmol of each probe mixture to 100 ul of probe hybridization buffer at 37°C.

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).

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.

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.)

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.

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.

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.

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)

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).

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

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.

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.

DAY 6

Wash embryos with 5xSSCT three times for 2 hours.

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.

DAY 7

Wash with 5xSSCT twice for 2 hours.

Incubate embryos in 1:2000 DAPI in 5xSSCT for 2 hours followed by 5xSSCT wash for 5 minutes.

Incubate embryos in Fructose-Glycerol clearing as described above before imaging.