TITLE: Hybridization Chain Reaction (HCR) In Situ Protocol
AUTHORS: Heather Bruce, Gabby Jerz, Sophia  Kelly, Jenny  McCarthy, Aaron Pomerantz, Gayani Senevirathne, Alice Sherrard, Dennis Sun, Carsten Wolff, Nipam Patel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Hybridization Chain Reaction (HCR) In Situ Protocol</span></div></div><br/><div class = "text-block"><span>This protocol largely follows the HCR v3.0 protocol for whole-mount Drosophila embryos (Choi et al. 2018) with a few adaptations that simplify the procedure and improve signal to noise ratio in our hands. Our lab has used it with good success in </span><span style = "font-style:italic;">Drosophila melanogaster</span><span> (fruit fly) embryos and imaginal discs, </span><span style = "font-style:italic;">Junonia coenia</span><span> (butterfly) embryos and imaginal discs, </span><span style = "font-style:italic;">Antheraea polyphemus</span><span>, (moth) pupal wings, </span><span style = "font-style:italic;">Parhyale hawaiensis</span><span> (amphipod crustacean) embryos, </span><span style = "font-style:italic;">Tribolium castaneum </span><span>(beetle) embryos, </span><span style = "font-style:italic;">Artemia franciscana</span><span> (brine shrimp) nauplii and adults, </span><span style = "font-style:italic;">Acanthoscurria geniculata</span><span> (tarantula) embryos, </span><span style = "font-style:italic;">Daphnia magna </span><span>(water flea) embryos, and </span><span style = "font-style:italic;">Danio rerio</span><span> (zebrafish) embryos. For some examples, please refer to Bruce & Patel 2020 and images below.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Fixation:</span></div><div class = "text-block"><span>Dissect tissue and fix for 10-40 minutes as you would for standard in situ or antibody staining. For fixation, we generally use 3.2% paraformaldehyde mixed in 1X artificial seawater (for </span><span style = "font-style:italic;">Artemia</span><span> and</span><span style = "font-style:italic;"> Parhyale</span><span>) or 1X PBS (for </span><span style = "font-style:italic;">Junonia</span><span>, </span><span style = "font-style:italic;">Antheraea</span><span>, and </span><span style = "font-style:italic;">Anthoscurria</span><span>). For </span><span style = "font-style:italic;">Drosophila </span><span>and </span><span style = "font-style:italic;">Tribolium</span><span>, a standard heptane/PEM-FA fixation, with subsequent methanol devitillenization is followed (detailed protocol in Patel 1994). We routinely dehydrate the embryos/tissue into 100% methanol after fixation and store the samples at -20°C for several years without a problem. This dehydration in methanol is not strictly necessary, but often makes the samples less prone to floating in later steps of the protocol. We no longer carry out the xylene treatment or ProteinaseK digestion steps found in other protocols.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Probes:</span></div><div class = "text-block">We have had our probes synthesized by Molecular Instruments (Los Angeles, CA. USA). They recommend starting with a minimum of 1.4kb of sequence with 20 probe sets created from this. We have had success with shorter sequences – our shortest has been 777bp with just a 9 probe set, but obviously longer is better.</div><br/><div class = "text-block">We have used five different hairpin amplifier colors (Alexa 488, 514, 546, 594, and 647) available from Molecular Instruments, and all have worked well. We routinely carry out three color in situs using the combination of Alexa 488, 546, and 647. Additionally we have had success with five color labeling experiments with up to nine probes. To create clear separation of five color channels, spectral imaging and linear unmixing have been necessary.</div><br/><div class = "text-block">For some species, we have had issues with autofluorescence at shorter wavelengths. In these cases we have found that the Alexa 488 hairpins have led to a poor signal to noise ratio, and favor using the longer wavelength probes (546, 594, and 647). </div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dz7ibid47.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dz7nbid47.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dz7sbid47.jpg" /></div><br/><div class = "text-block">References:</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Choi HMT, Schwarzkopf M, Fornace ME, Acharya A, Artavanis G, Stegmaier J, Cunha A, Pierce NA. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development. 2018 Jun 26;145(12):dev165753. doi: 10.1242/dev.165753. PMID: 29945988; PMCID: PMC6031405.</li><li style = "counter-reset:ol0;">Rehm EJ, Hannibal RL, Chaw RC, Vargas-Vila MA, Patel NH. Fixation and dissection of Parhyale hawaiensis embryos. Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.prot5127. doi: 10.1101/pdb.prot5127. PMID: 20147022.   </li><li style = "counter-reset:ol0;">Patel, N H. “Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes.” Methods in cell biology vol. 44 (1994): 445-87. doi:10.1016/s0091-679x(08)60927-9.                             </li><li style = "counter-reset:ol0;">Bruce HS, Patel NH. Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments. Nat Ecol Evol. 2020 Dec;4(12):1703-1712. doi: 10.1038/s41559-020-01349-0. Epub 2020 Dec 1. PMID: 33262517.</li></ol></div></div>

[STEPS]
?. [Day 1 ]
Perform PTw Washes:Note: If your embryos/tissue are stored in methanol, first rehydrate into PTw. For larger embryos, we recommend rehydrating step-wise 75/50/25% methanol in PTw).                1 × 10min     PTw wash                1 × 5min       PTw wash (Sonicate here if necessary due to cuticle, see Step 18)                1 × 5min       PTw wash
?. [Day 1 ]
Permeabilize in 300-500µL Detergent Solution for 30min at room temperature.During this step, pre-warm probe hybridization buffer to 37°C (200µL/tube).
?. [Day 1 ]
Pre-hybridize embryos in pre-warmed 200µl of probe hybridization buffer for 30min at 37°C.                1 X 30min     Probe Hybridization Buffer Note for reusing probe: Probe is presumably already in correct concentration, pre-warm to 37°C, then skip to Step 5. Number of reuses will depend on sample type and concentration used.
?. [Day 1 ]
Prepare probe solution by adding 0.8pmol (0.8µl of probe from 1uM stock solution) of each probe mixture to 200µL of probe hybridization buffer at 37°C. Troubleshooting tip: If signals are weak, try increasing probe concentration up to 2-3x. We have found this increased probe concentration to be useful when dealing with shorter than recommended starting sequences (fewer probe sets).
?. [Day 1 ]
Remove the pre-hybridization solution and add the probe solution.
?. [Day 1 ]
Incubate embryos overnight (12-16h) at 37°C. Troubleshooting tip: Extending incubation period up to 20 hours and/or increasing probe concentration up to 5-fold may be useful, especially with probes that have less than 20 probe pairs.
?. [Day 2]
Remove and SAVE PROBE SOLUTION, which can be reused at least 3 times. Save used probe solutions at -20°C.
?. [Day 2]
Wash samples 4 × 15min with 1mL of pre-warmed probe wash buffer at 37°C.                 1 × 15min    Probe Wash Buffer                 1 × 15min    Probe Wash Buffer                 1 × 15min    Probe Wash Buffer                 1 × 15min    Probe Wash Buffer
?. [Day 2]
Wash samples 2 × 5min with 1mL of 5X SSCT at room temperature.                1 × 5min    5x SSCT                 1 × 5min    5x SSCT
?. [Day 2]
Pre-amplify embryos with 1mL of pre-equilibrated amplification buffer for 30min at room temperature.Make sure to pre-equilibrate amplification buffer to room temperature before use.                1 × 30min    Amplification Buffer
?. [Day 2]
During the pre-amplification step, prepare hairpins. Mix 2µL (3µM stock) of each hairpin h1 and 2µL of each hairpin h2 in 100µL of amplification buffer at 95°C for 90sec, then cool to room temperature in a dark drawer for 30min. Troubleshooting tip: Doubling hairpin concentration can help to boost signal.Note: If reusing hairpins from prior experiment, heat and cool, then skip to Step 13.
?. [Day 2]
Remove the pre-amplification solution and add the hairpin solution.
?. [Day 2]
Incubate the embryos overnight (2–16h) in the dark at room temperature.
?. [Day 3]
SAVE HAIRPIN MIXTURE. Can be reused multiple times. Save used hairpin mixtures at -20°C.Remove excess hairpins by washing with 1mL of 5X SSCT at room temperature:                 1 × 5min    5X SSCT                 1 × 5min    5X SSCT                 1 × 30min    5X SSCT                 1 × 30min    5X SSCT                 1 × 5min    5X SSCT
?. [Day 3]
Incubate embryos in 50% glycerol solution (in 1X PBS) with DAPI (skip if DAPI is not needed):                30min-1h if using 1.0µg/mL DAPI                2 h or overnight at 4°C if using 0.1µg/mL DAPI
?. [Day 3]
Replace DAPI glycerol with 50%-70% glycerol (in 1X PBS) and store at 4°C. Troubleshooting tip: 1X PBS must be at pH 7.40, deviation from this may result in rapid loss of signal.
?. [Notes]
If the sample has substantial cuticle that would normally block penetration of probes (such as Artemia), we follow the sonication procedure described in Patel 1994, as indicated in Step 1.
?. [Notes]
If you have problems with changing the solutions because the embryos do not settle, fold then twist a piece of Nitex mesh over a glass Pasteur pipette to make a filter, secure twisted Nitex with a ring of cut plastic transfer pipette, and use this to remove buffer without sucking up embryos.
?. [Notes]
You will note that there is no ProteinaseK treatment step in this protocol. Many years ago, when working with grasshoppers (and more standard DIG-RNA probes), we found that this ProteinaseK treatment time had to be optimized for each stage of development, and led to some degradation in morphology. We modified the DIG in situ protocol to instead use a Detergent Treatment step. In adapting the HCR protocol, we have continued with the Detergent step (Step2), and left out ProteinaseK treatment. Since there is no ProteinaseK treatment, a post-fixation step is unnecessary. Indeed, in our tests, we found that a post-fixation step (after Detergent treatment) results in a lower signal to noise ratio. Finally, addition of 0.1% SDS to the hybridization buffer was tested and deemed unnecessary.
?. [Day 2]
Pre-heat probe wash buffer to 37°C before use, equilibrate amplification buffer to room temperature before use, and pre-set heat block to 95°C.
?. [Notes]
If you would like to combine in situ and antibody staining, we have had good success with starting a standard immunofluorescence staining protocol following step 15 of this protocol.