HCR on Whole Mouse Embryos

Whole-mount mouse embryo HCR protocol adapted from Molecular Instruments V3.0 protocol Read the original protocol for appropriate cautions regarding buffers contained in the HCR kit (https://files.molecularinstruments.com/MI-Protocol-HCRv3-Mouse-Rev6.pdf) or Choi et al 2020¹

Day 1

1. Fix dissected embryos overnight in 4% PFA, 4°C while rocking.

Day 2

1. Dehydrate gradually into 100% Methanol (PBT -> 25% methanol/PBT -> 50% methanol/PBT -> 75% methanol/PBT -> 2 X 100% methanol), store embryos at -20°C until needed.

Note: Embryos can continue be used for HCR following a minimal period of 1-2 hours in methanol (methanol is required for proper tissue fixation).

Day 3

1. Rehydrate gradually into PBT (75% methanol/PBT -> 50% methanol/PBT -> 25% methanol/PBT -> PBT). 
2. Bleach embryos in 6% hydrogen peroxide in PBS for 15-60 minutes depending on stage (longer for older embryos).
                         1 mL    5 mL      10 mL  
   50% H₂O₂     0.12 mL 0.6 mL  1.2 mL
   PBT                0.88 mL 4.4 mL  8.8 mL
    
3. Wash embryos twice with PBT for 5 minutes per wash while rocking.
4. Digest embryos with proteinase K at room temp (10 µg/mL PBT) without rocking.
   Note: We have found that digestion times that are ~25% longer than we would typically use for chromogenic in situ hybridization work better. For example, we would normally digest E9.5 whole embryos for 8 minutes for chromogenic in situ hybridization, so would digest for 10 minutes for HCR.
5. Wash embryos twice with PBT for 5 minutes per wash without rocking.
6. Fix embryos with 4% PFA for 20 minutes at room temperature while rocking.
7. During fixation step, preheat Hybridization Buffer (V3.0, 30% Formamide) to 37°C
8. Wash three times with PBT for 5 minutes per wash while rocking.
9. Make a 1:1 solution of Hybridization Buffer and PBT and wash embryos in 1:1 solution rocking, for 5-10 minutes or until the embryos equilibrate. 
   Note: The Hybridization Buffer is very viscous and this step is desiged help gently equilibrate embryos.
10. Wash 5-10 minutes at room temperature in warmed (37°C) Hybridization Buffer.
11. Replace Hybridization Buffer from step 10 with fresh Hybridization Buffer and incubate at 37°C for minimally 30 minutes while rocking. 
    Note: We typically incubate embryos for 1-3 hours in Hybridization Buffer.
12. Make probe solution by adding 2 µL of each probe (probe stock 1 µM) to 500 µL of prewarmed 37°C Hybridization Buffer (final concentration 4 nM of each probe).
    Note: Mix all probes in the same tube however ensure initiator sequences are unique between probes.
13. Replace Hybridization Buffer with Probe Solution and incubate at 37°C overnight while rocking.

Day 4

1. Warm Probe Wash Buffer to 37°C.
2. Remove and save probe solution, store at -20°C. 
   Note: We have reused probes numerous times with smaller embryos (E9.5 and younger) without discernable loss of signal, however with older embryos signal does decline with repeated use.
3. Perform a quick rinse with Probe Wash Buffer then wash three times at 37°C for 20 minutes each while rocking. 
4. Rinse with 5x SSCT, then wash three times at room temperature for 5 minutes each while rocking. 
5. Meanwhile bring Amplification Buffer to room temperature. 
6. Make a 1:1 solution of Amplification Buffer and PBT, and wash embryos for 10 minutes at room temperature while rocking.
7. Equilibrate embryos in Amplification Buffer at room temperature for 30 minutes while rocking.
   Note: This incubation can be longer than 30 minutes.
8. Meanwhile melt and snapcool hairpins:
   1. Choose hairpins that have same initiator as probe to be detected and desired fluorophore. 
   2. Each hairpin has two components, H1 and H2, which must be kept separate before snap-cooling to prevent premature polymerization.
   3. Place 20 µL of each hairpin component in separate PCR tubes (ie. 20 µL of H1 into a tube and 20 µL of H2 into a separate tube), then melt for 90 seconds at 95°C in a PCR thermalcycler.
   4. Remove the tubes from the thermalcycler after the 90 seconds and place in a dark drawer for 30 minutes to allow the oligos to refold and form hairpin structures.
   5. Mix all hairpins together in 500 µL of room temperature Amplification Buffer and invert the tube to mix (ensure solution is well mixed).
9. Remove Amplification Buffer from embryos and apply enough hairpin containing Amplification Buffer to cover the embryos and gently flick to mix while ensuring embryos remain in the solution. 
   Note: We typically use 125 µL of Amplification Buffer with hairpin per tube of E7.5-10.5 embryos, but use higher volume for older embryos (want to achieve a minimal 2-3x the volume of hairpin to embryo volume).
10. Rock embryos overnight at room temperature protected from light.
    Note: We generally used a styrofoam rack that has been cut to allow the tubes to lay at ~45 degree angle then cover with foil to hold the tubes in place and protect from light. 

Day 5

1. Peform a quick wash of the embryos with 5x SSCT to remove hairpin solution, then wash three times with 5x SSCT for 5 minutes each rocking at room temperature while protected from light.
2. Ensure HCR worked by checking briefly on a fluorescent stereoscope, then soak in DAPI solution (0.5 µg/mL Dapi in 5x SSC with 0.1% Triton X-100 and 1% Tween20) overnight at room temperature while rocking. 
   Note: If Dapi counterstaining is not required one may proceed to the mouting and clearing step after approximately 30 minutes of additional washing in 5x SSCT.

Day 6

1. Rinse with 5x SSCT, then embed in Ultralow gelling temperature agarose (Sigma A5030) that has been melted in a 65°C water bath or microwave then cooled to room temperature.
   Note: It is important to use Ultralow gelling temperature agarose to ensure agarose temperature is at or below probe and hairpin annealing temperature (37°C and 25°C respectively).
   Note: Ultralow gelling agarose requires placement on ice to fully gel. 
2. Clear embryos after embedding for imaging (ie. Confocal Microscopy). 
   Note: We generally use Ce3D² for clearing however have also successfully used ScaleA2³.

Additonal Notes: Embryos typically retain fluorescence for at least several weeks before clearing when kept protected from light. 

Solutions

4% paraformaldehyde

1 g                       PFA Powder

25 mL                 PBS

50 µL                  10N Sodium Hydroxide

 *heat at 65°C until powder is dissolved, then aliquot and freeze.

PBT

0.5 mL                Tween-20

500 mL              PBS

Hybridization Buffer

-see original protocol on the Molecular Instruments website: https://files.molecularinstruments.com/MI-Protocol-HCRv3-Mouse-Rev6.pdf

Amplification Buffer

-see original protocol on the Molecular Instruments website https://files.molecularinstruments.com/MI-Protocol-HCRv3-Mouse-Rev6.pdf

Probe Wash Buffer

-see original protocol on the Molecular Instruments website https://files.molecularinstruments.com/MI-Protocol-HCRv3-Mouse-Rev6.pdf

5x SSCT

125 mL              20x SSCT

0.5 mL                Tween 20

374.5 mL           deionized/RO H₂O

1.  Choi, H.M.T., Schwarzkopf, M. & Pierce, N.A. Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount Vertebrate Embryos: qHCR and dHCR Imaging (v3.0). Methods Mol Biol 2148, 159-178 (2020).

2.  Anderson, M.J., Magidson, V., Kageyama, R. & Lewandoski, M. Fgf4 maintains Hes7 levels critical for normal somite segmentation clock function. Elife 9 (2020).

3.  Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14, 1481-1488 (2011).

Category