May 10, 2022
Hybridization Chain Reaction combined with Immunohistochemistry for Whole-Mount Embryos
- Ali M Elagoz1,
- Ruth Styfhals1,
- Sofia Maccuro1,
- Luca Masin2,
- Lieve Moons2,
- Eve Seuntjens1
- 1Research group of Developmental Neurobiology, KU Leuven;
- 2Research group of Neural Circuit Development and Regeneration, KU Leuven
- Developmental Neurobiology Seuntjens Lab
Protocol Citation: Ali M Elagoz, Ruth Styfhals, Sofia Maccuro, Luca Masin, Lieve Moons, Eve Seuntjens 2022. Hybridization Chain Reaction combined with Immunohistochemistry for Whole-Mount Embryos. protocols.io https://dx.doi.org/10.17504/protocols.io.bxz6pp9e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: September 07, 2021
Last Modified: May 10, 2022
Protocol Integer ID: 53022
Keywords: HCR, hybridization chain reaction, wholemount, immunohistochemistry, HCR v3.0, cephalopod, octopus
Abstract
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.
Materials
BUFFER RECIPES FOR IN SITU HCR v3.0
Probes, amplifiers, probe hybridization buffer and probe wash buffer should be stored at -20°C.
Amplification buffer should be stored at 4°C.
Keep these reagents on ice at all times during probe and amplifier preparation. Make sure all solutions are well mixed before use.
Buffer recipes for sample preparation
4% paraformaldehyde (PFA) For 25 mL of solution
4% PFA 1 g of PFA powder
1x PBS 25 mL of 1x PBS
Heat solution at 50–60 °C to dissolve the powder
PBST For 50 mL of solution
1x PBS 5 mL of 10x PBS
0.1% Tween 20 500 uL of 10% Tween 20
Fill up to 50 mL with ultrapure H2O
Proteinase K solution For 2 mL of solution
10 ug/mL proteinase K 1,08 uL of 20 mg/mL proteinase K
Fill up to 2 mL with PBST
Probe hybridization buffer (aliquot and store at -20°C) => Prepare in 50 ml falcon
Final concentration For 40 ml
30% formamide 12 ml formamide
5x SSC 10 ml of 20 x SSC
9 mM citric acid pH 6 360 ul 1 M citric acid pH 6
0,1% Tween 20 400 ul of 10% Tween 20
50 ug/ml heparin 200 ul of 10 mg/ml heparin
1 x Denhardts solution 800 ul of 50x Denhardt’s solution
10% dextran solution 8 ml of 50% dextran sulfate
Fill up to 40 ml with ultrapure H20
Probe wash buffer (100 ml per experiment/ freeze in 50 ml aliquots) Store at -20°C => Prepare in 0.5l bottle
Final concentration For 400 ml
30% formamide 120 ml formamide
5x SSC 100 ml of 20 x SSC
9 mM citric acid pH 6 3,6 ml of 1 M citric acid pH 6
0.1% Tween 20 4 ml of 10% Tween 20
50 ug/ml heparin 2 ml of 10 mg/ml heparin (4°C)
Fill up to 400 ml with ultrapure H20 (170,4 mls)
Amplification buffer (4°C) - prepare in 50 ml falcon - make 10 ml aliquots
Final concentration For 40 ml
5 x SSC 10 ml of 20 x SSC
0,1% Tween 20 400 ul of 10% Tween 20
10% Dextran sulfate 8 ml of 50% Dextran sulfate
Fill up to 40 ml with ultrapure H20
5x SSCT (RT) - prepare 1 L
Final concentration For 1 L
5x SSC 250 ml of 20 x SSC
0,1% Tween 20 10 ml of 10% Tween 20
Fill up to 1L with ultrapure H20 (740 ml)
50% Dextran sulfate => best to dissolve overnight (add water gradually) - prepare in falcon
20 g of dextran sulfate powder
Fill up to 40 ml with ultrapure H20.
=> make aliquots of 8 ml and store at -20°C.
10 mg/ml Heparin (50 ml) => aliquot per 10 ml store at 4°C
500 mg heparin
Fill up to 50 ml with ultrapure H20.
Filter 0.2 uM and store at 4°C.
! store powder at room T, store solution at 4°C.
1M citric acid pH 6 (50 ml)
10.5 g in 30 ml
adjust pH to 6.0 with NaOH : make 20 ml of conc NaOH in Rnase-Dnase free water, let dissolve and add slowly.
Fill up to 50 ml
Hairpins
Ordered from MI:
B1 546 600 pmol
B2 647 600 pmol
B3 488 600 pmol
300 pmol (100 ul of 3 pmol/ul = 3 uM)
Aliquot per 15 ul and store at -20°C.
Probes
Ordered DNA oPools from IDT (without 5’ mod): 50 pmol
Dissolved in 100 ul ultrapure water (or Tris pH 7.5). Final concentration is 0.5 pmol/ul. Aliquot per 10 ul and store at -20°C. We use 0.3 pmol = 0.6 ul per slide.
Reagents and supplies
Heparin (Sigma cat H3393): powder stored at room T, aliquots are stored at 4°C.
20x Sodium Chloride Sodium Citrate (SSC: Invitrogen) 1 L, stored at room T.
10 % Tween 20 (Bio-Rad). Protect from light. Stored at Room T.
50x Denhardt’s solution (Aliquot and store at -20°C)
Dextran sulfate (Sigma cat D8906): powder stored at 4°C, aliquots are stored at -20°C.
Safety warnings
Formamide is toxic substance so make sure to take the appropriate measures.
Embryo Fixation and Preparation
Embryo Fixation and Preparation
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.
Dehydration
Dehydration
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.
Rehydration and Permeabilization
Rehydration and Permeabilization
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.
Hybridization
Hybridization
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).
Wash Steps
Wash Steps
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.)
Amplification
Amplification
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).
Wash Steps and Imaging
Wash Steps and Imaging
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.
Immunohistochemistry (IHC)
Immunohistochemistry (IHC)
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.