Hybridization Chain Reaction (HCR) In Situ Protocol v1
Authors: S Bruce Heather , Jerz Gabby , R Kelly Sophia , McCarthy Jenny , Pomerantz Aaron , Senevirathne Gayani , Sherrard Alice , A Sun Dennis , Wolff Carsten , H Patel Nipam 
Published: July 2021

Hybridization Chain Reaction (HCR) In Situ Protocol 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 Drosophila melanogaster (fruit fly) embryos and imaginal discs, Junonia coenia (butterfly) embryos and imaginal discs, Antheraea polyphemus, (moth) pupal wings, Parhyale hawaiensis (amphipod crustacean) embryos, Tribolium castaneum (beetle) embryos, Artemia franciscana (brine shrimp) nauplii and adults, Acanthoscurria geniculata (tarantula) embryos, Daphnia magna (water flea) embryos, and Danio rerio (zebrafish) embryos. For some examples, please refer to Bruce & Patel 2020 and images below. Fixation: 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 Artemia and Parhyale) or 1X PBS (for Junonia, Antheraea, and Anthoscurria). For Drosophila and Tribolium, 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. Probes: 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. 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. 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). References: 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. 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. 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. 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.