TITLE: Hybridization chain reaction (HCR) protocol for tails of mouse embryos
AUTHORS: Paul Gerald Layague Sanchez, Hidenobu Miyazawa, Molecular Instruments

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for the in situ hybridization chain reaction (HCR) of intact tails (somites and presomitic mesoderm, PSM) of mouse embryos. This is adapted from the in situ HCR v 3.0 protocol for whole-mount mouse embryos available in 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 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
200 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
?. [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 ]
Fix samples in 4% formaldehyde ( 37% formaldehyde diluted to  with PBST) for  at . Pre-warm probe hybridization (PH) buffer at  for later use.
2.16 ml
20 ml
0 Room temperature
37 °C
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 .
Probe hybridization (PH) buffer contains formamide, which is toxic.
?. [Day 1: Recovery and fixation of samples ]
Recover samples, wash quickly with cold PBS, and fix in 4% formaldehyde ( 37% formaldehyde diluted to  with PBS). Keep in formaldehyde overnight (around ) at  in the cold room, with gentle shaking/rolling.
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 ]
Put fresh PH buffer to the tubes and incubate for  at . Meanwhile, prepare probe solution by adding 2 pmol ( of stock) of odd probe mixture and 2 pmol of even probe mixture to every  of pre-warmed PHP buffer (from Step 8).
37 °C
2 µl
500 µl
Probe hybridization (PH) buffer contains formamide, which is toxic.
?. [Day 3: Rehydration of samples and HCR detection stage ]
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 , and equilibriate amplification buffer to .
37 °C
0 Room temperature
Probe wash (PH) buffer contains formamide, which is toxic.
?. [Day 4: HCR amplification stage ]
Wash samples with probe wash buffer at . Wash 1/4 Wash 2/4 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 (30 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 ]
In separate tubes, for every  hairpin mixture, prepare 30 pmol ( of  stock or  of  stock) of hairpin h1 (tube 1) and 30 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
15 µl
10 µl
95 °C
0 Room temperature
?. [Day 4: HCR amplification stage ]
Remove the amplification buffer from the sample tubes, and add the hairpin mixture. Incubate overnight (around  ) in the dark at .
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 (:  of  DAPI diluted to  with 5x SSCT) in the dark at .Wash + DAPI 1/3 Wash + DAPI 2/3 Wash + DAPI 3/3
20 µl
20 ml
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.