TITLE: Hybridization chain reaction (HCR) protocol for Gastruloids (ESC aggregates)
AUTHORS: Stefano Vianello, Jisoo Park, Matthias Lutolf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for the in situ hybridization chain reaction (HCR) of Gastruloids (3D aggregates of mouse embryonic stem cells). This is an adaptation of the same protocol as applied to mouse tails (Sanchez et al; </span><a href="https://dx.doi.org/10.17504/protocols.io.7pyhmpw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.7pyhmpw</span></a><span style = ":UNDERLINE;">) </span><span>in turn adapted from the whole-mount mouse embryo protocol available on the website of Molecular Instruments (for details, please follow this </span><a href="https://www.molecularinstruments.com/protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">link</span></a><span>).</span></div><div class = "text-block">References</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Choi, H., Schwarzkopf, M., Fornace, M., Acharya, A., Artavanis, G., & Stegmaier, J. et al. (2018). Third-generationin situhybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</span><span style = "font-style:italic;">Development</span><span style = "font-style:italic;font-style:italic;">,</span><span style = "font-style:italic;">145 </span><span>(12), dev165753. doi: </span><a href="http://dx.doi.org/10.1242/dev.165753" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1242/dev.165753</span></a></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Choi, H., Calvert, C., Husain, N., Huss, D., Barsi, J., & Deverman, B. et al. (2016). Mapping a multiplexed zoo of mRNA expression.</span><span style = "font-style:italic;">Development</span><span style = "font-style:italic;font-style:italic;">,</span><span style = "font-style:italic;">143 </span><span>(19), 3632-3637. doi: </span><a href="http://dx.doi.org/10.1242/dev.140137" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1242/dev.140137</span></a></li></ul></div></div>

[STEPS]
?. [Day 1: Recovery and fixation of samples ]
Precoat a well of a 6well plate with 2mL serum solution (e.g. PBSFT: PBS+10%FBS+0.2%Triton-X100), for around 30min. Remove the serum solution and replace with with 2mL 4% formaldehyde ( 37% formaldehyde diluted to  with PBS).To recover samples, use a cut P1000 pipette to pick up each Gastruloid from its well in the 96well plate, collecting all Gastruloids in a 15mL Falcon tube. Once Gastruloids have sedimented at the bottom of the tube, use a glass pipette connected to a vacuum line to remove as much medium as possible.Wash quickly by adding 2mL PBS, and transfer all Gastruloids to the pre-prepared PFA-filled well.Keep in formaldehyde overnight (around ) at  in the cold room, with gentle shaking.
1.08 mL
10 mL
4 °C
Formaldehyde is toxic. Operate under a fume hood and wear appropriate hand, body, and eye protection.
To reduce autofluorescence, it is recommended to use fresh fixative and PBS with no divalent cations (like Ca2+ and Mg2+).
?. [Day 2: Dehydration of samples ]
Wash samples with PBST (0.1% Tween20 in 1x PBS).Wash 1/2 Wash 2/2
?. [Day 2: Dehydration of samples ]
Dehydrate samples (one sample per tube) with a series of graded methanol : PBST (PBS + 0.1% Tween) washes.25% methanol : 75% PBST 50% methanol : 50% PBST 75% methanol : 25% PBST 100% methanol 100% methanol
?. [Day 2: Dehydration of samples ]
Incubate samples at  overnight (around ) or until use.
-20 °C
?. [Day 3: Rehydration of samples and HCR detection stage ]
Rehydrate samples (one sample per PCR tube, capacity =  max) with a series of graded methanol : PBST (PBS + 0.1% Tween) washes.100% methanol 75% methanol : 25% PBST 50% methanol : 50% PBST 25% methanol : 75% PBST 100% PBST 100% PBST
500 µl
?. [Day 3: Rehydration of samples and HCR detection stage ]
Treat samples with  proteinase K (Merck, CAS # 38450-01-6) for  at .
0 Room temperature
Proteinase K is sold at a concentration of   . Make a   solution by diluting   of Proteinase K in   PBST.Use 1:2 by removing  half of the volume from the tube with the samples, and replacing with this new Proteinase K solution. The resulting concentration of Proteinase K in the tube will be of
?. [Day 3: Rehydration of samples and HCR detection stage ]
Wash samples with PBST.Wash 1/2 Wash 2/2
?. [Day 3: Rehydration of samples and HCR detection stage ]
Refix samples in 4% formaldehyde ( 37% formaldehyde diluted to  with PBST) for  at . Pre-warm probe hybridization (PH) buffer at  for later use ( /sample).
2.16 mL
20 mL
0 Room temperature
37 °C
1.5 mL
Formaldehyde is toxic. Operate under a fume hood and wear appropriate hand, body, and eye protection.
Probe hybridization (PH) buffer contains formamide, which is toxic.
To reduce autofluorescence, it is recommended to use fresh fixative and PBS with no divalent cations (like Ca2+ and Mg2+).
?. [Day 3: Rehydration of samples and HCR detection stage ]
Wash samples with PBST.Wash 1/3 Wash 2/3 Wash 3/3
?. [Day 3: Rehydration of samples and HCR detection stage ]
Wash samples with PH buffer for . To aid later buffer changes, bring Gastruloids to the bottom of the tube by centrifuging  , or until Gastruloids pellet to the bottom
Centrifuge: 1500 34, 1 min
Probe hybridization (PH) buffer contains formamide, which is toxic.
?. [Day 3: Rehydration of samples and HCR detection stage ]
Put fresh PH buffer to the tubes and incubate for  at . Meanwhile, prepare probe solution by adding 4 pmol ( of stock; since 2uM is 2pmol/uL) of odd probe mixture and 4 pmol of even probe mixture to every  of pre-warmed PHP buffer (from Step 8).
37 °C
2 µl
500 µl
We are using a reaction concentration of each HCR probe of 8 nM (8e-9 mol/L, 8e-12mol/mL, = 8pmol/mL )For a 500 µL reaction volume, each experiment will require 4 pmol of each probe.probe is sold as 1um = 1pmol/uL , so use 4 uL
Probe hybridization (PH) buffer contains formamide, which is toxic.
?. [Day 3: Rehydration of samples and HCR detection stage ]
Reserve part of your samples to a new tube, as negative controls. Remove PH buffer and add the probe solution. Incubate overnight (around ) at .
37 °C
?. [Day 4: HCR amplification stage ]
Pre-warm probe wash buffer at  ( /tube), and equilibriate amplification buffer to  ( /tube)
37 °C
2 mL
0 Room temperature
1 mL
Probe wash (PH) buffer contains formamide, which is toxic.
?. [Day 4: HCR amplification stage ]
In separate tubes, for every  hairpin mixture, prepare 48 pmol (of  stock; since 3uM stock is 3pmol/uL) of hairpin h1 (tube 1) and 48 pmol of hairpin h2 (tube 2). Snap-cool hairpins by heating the tubes at  for  and cool to  in the dark for at least .
500 µl
16 µl
95 °C
0 Room temperature
We are using a reaction concentration of each HCR amplifier hairpin of 96 nM. (96e-9 mol/L, 96e-12mol/mL, = 96pmol/mL . For a 500 µL reaction volume, each experiment will thus require 48 pmol of each hairpin.probe is sold as 3um = 3pmol/pL , so use 16 uL
?. [Day 4: HCR amplification stage ]
Wash samples with probe wash buffer at . Wash 1/4 Wash 2/4 (you can start step 14 during this wash)Wash 3/4 Wash 4/4
37 °C
Probe wash (PH) buffer contains formamide, which is toxic.
?. [Day 4: HCR amplification stage ]
Wash samples with 5x SSCT (0.1% Tween20) at . Wash 1/2 Wash 2/2
37 Room temperature
To prepare   of 5x SSCT, dilute   of 20x SSC and   Tween20 to    with distilled H2O.
?. [Day 4: HCR amplification stage ]
Wash samples with pre-equilibriated amplification buffer (Step 13) for  at . Meanwhile, prepare the hairpin mixture (48 pmol of each hairpin in  mixture) by mixing the snap-cooled hairpins h1 and h2 (Step 14) to amplification buffer at .
0 Room temperature
500 µl
0 Room temperature
?. [Day 4: HCR amplification stage ]
To aid sedimentation of the Gastruloids, centrifuge Remove the amplification buffer from the sample tubes, and add the hairpin mixture.Add DAPI  (1:500 dilution of a  stock)Incubate overnight (around  ) in the dark at .
Centrifuge: 1500 34, 30 s
0 Room temperature
For dHCR imaging, amplify for a shorter period of time to ensure single-molecule dots are diffraction-limited.
?. [Day 5: Washing, nuclear staining, and imaging]
Wash samples with 5x SSCT for  in the dark at .
0 Room temperature
?. [Day 5: Washing, nuclear staining, and imaging]
Stain nuclei with 1:1000 DAPI () in the dark at .Wash + DAPI 1/3 Wash + DAPI 2/3 Wash + DAPI 3/3
0 Room temperature
?. [Day 5: Washing, nuclear staining, and imaging]
Wash samples with 5x SSCT in the dark at Wash 1/2 Wash 2/2
0 Room temperature
?. [Day 5: Washing, nuclear staining, and imaging]
Samples are now ready for imaging. Prior to imaging, the samples could be cleared using the fructose-glycerol clearing solution described in Dekkers et al., 2019. Incubate samples in clearing solution in the dark at  for at least .
4 °C
To prepare the fructose-glycerol clearing solution, dissolve   fructose in   glycerol and   water on a magnetic stirrer (start the dissolution at least a day before intended day of clearing because it will take a while for fructose to dissolve). The clearing solution can be stored in the dark at   for at most 1 month. This solution can be used to mount the sample on microscope slide/cover glass. Take note that the clearing is solution has high viscosity.
?. [Day 5: Washing, nuclear staining, and imaging]
Mount the Gastruloids by using a cut P1000 pipette and transferring them to a  drop of Fluoromount-G. Cover with a #1 coverslip and wick excess liquid from the sides by using a Kimtech wipe.Seal all edges with Permount
35 µl
?. [Probe preparation]
#Note: Steps 1 and 2 only apply if making/ordering your own probes. If buying premade ones from Molecular Instruments, they will come already mixed together and resuspended. Skip to step 3.Resuspend your probes:order each probe at the minimal amount (e.g. 25nmol), Desalted, and DryUpon receipt, spin each tube (e.g. 6000g centrifuge) to collect the pellet to the bottom of the vialAdd the correct amount of DNAse-free water  Vortex briefly to mix the probes well into solutionRe-centrifuge to collect all the solution away from the capStore at -20C long termVortex briefly to mix the probes well into solutionRe-centrifuge to collect all the solution away from the capStore at -20C long term
To standardise the volumes required in the protocol below, aim to resuspend your probes to X* 20uM, where X=number of primer pairs. As such, if you designed 10 probe pairs, you will resuspend each probe to 10*20=200uM
?. [Probe preparation]
Prepare ODD and EVEN mixes:in a DNAse-free 0.5mL Eppendorf tube, collect 1uL of each ODD probedilute 1:10 by adding 9 volumes of DNAse-free wateron a separate DNAse-free 0.5mL Eppendorf tube, collect 1uL of each EVEN probedilute 1:10 by adding 9 volumes of DNAse-free water Store at -20C long termIndependently on the number of primers designed, these ODD and EVEN mixes both have a concentration of 2uMon a separate DNAse-free 0.5mL Eppendorf tube, collect 1uL of each EVEN probedilute 1:10 by adding 9 volumes of DNAse-free water Store at -20C long termIndependently on the number of primers designed, these ODD and EVEN mixes both have a concentration of 2uMStore at -20C long termIndependently on the number of primers designed, these ODD and EVEN mixes both have a concentration of 2uM
By mixing the same volume of each resuspended probe(1uL in this case), the resulting concentration of each probe in the mix will be 1/X of the original one, where X is the number of probes mixed. Because the original probes had been resuspended to X*20uM, the final concentration of each prob in this new mix is 20uM. By adding 9 volumes of DNAse-free water, the final concentration of each probe in the probe mix is now 2uM.Worked example:I bought 10 probe pairs, and resuspended each to 10*20uM = 200uM, as per Step 1I now take 1uL of each of the 10 ODD probes, and collect them in the same tubeThe resulting mix has a volume of 10*1uL = 10uL, and the concentration of each probe is now (1/10)*200uM = 20uMI add 9 volumes (9*10uL) of DNAse-free water. The resulting mix has a volume of 100uL, and each probe is now 2uMI repeat the same process with the EVEN probes