HCR RNA-FISH: simple, scalable, superior in situ hybridization. Free HCR RNA-FISH Starter Kits for the Pain Researcher community!

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HCR RNA-FISH: simple, scalable, superior in situ hybridization. Free HCR RNA-FISH Starter Kits for the Pain Researcher community!

In situ hybridization is an invaluable tool for neuroscience research, allowing researchers to visualize anatomical distribution and localization of DNA and RNA targets. However, given the complex nature of tissues in the nervous system, existing in situ hybridization methods are unable to perform robust analyses of neural gene expression, especially in thick, autofluorescent tissue samples. At Molecular Instruments (MI), our goal is to make in situ hybridization routine and accessible in any sample type, regardless of thickness or preparation. Our team designs and synthesizes kits for multiplexed, quantitative, high-resolution bioimaging for academic research, drug development, and clinical pathology/diagnostics.

Our technology, Hybridization Chain Reaction (HCR), is a next-generation signal amplification platform that enables 1-step multiplexed, isothermal, enzyme-free labeling of biomolecules. Originally developed in the Pierce Lab at Caltech in 2010, our HCR imaging platform is constantly improving to overcome multi-decade technological challenges in multiplexed, quantitative bioimaging.

HCR RNA fluorescence in situ hybridization (FISH) enables researchers to visualize the expression of multiple RNA targets simultaneously in the spatial context of the intact nervous system. We have also recently extended the benefits of HCR-based amplification to immunohistochemistry (IHC) (Schwarzkopf et al., 2021), allowing for accurate and precise protein quantitation with subcellular resolution.

HCR RNA-FISH and HCR IHC products enable simultaneous labeling of up to 10 RNA and protein targets using a single, unified amplification platform. This results in a method that is simpler, more scalable, and superior to any alternative available today.

Key Features:

  • Straightforward Multiplexing
    HCR enables 1-step parallel signal amplification of all targets simultaneously. Users can multiplex up to 10 targets! B6 - B10 amplifier channels are now available to order as custom amplifiers. If interested, please specify the amplifier channel you’d like in the comments field on the ordering interface.

  • No Custom Design Fees
    We know that custom fees can deter researchers from purchasing key research tools – that’s why we don’t charge any design fees to make our technology accessible to scientists around the world! One of our core values at MI is supporting our users in pursuing fundamental and groundbreaking research, so we design custom probes for any target RNA at no additional cost.

  • Deep Sample Penetration
    Existing in situ hybridization techniques either require extensive sample pretreatments to facilitate probe diffusion into thick tissue samples or cannot accommodate thick sample types at all. In contrast, HCR reagents are engineered to be small so that they can deeply penetrate into all sample types including thick tissue and whole-mount samples.

  • Compatible with Any Primary Antibody
    Have a preferred primary antibody that you have validated? Great! Bring your favorite primary antibodies for detection of any protein in combination with validated HCR secondary antibody probes. See how we conducted simultaneous multiplexed protein and RNA imaging on a 5 µm FFPE mouse brain section here: Scharzkopf et al., 2021

  • Compatible with Tissue Clearing
    HCR RNA-FISH is compatible with a variety of different clearing techniques. Both pre-HCR clearing (e.g., PACT, SHIELD) and post-HCR clearing (e.g., TDE) preserve RNA and are compatible with HCR:

Protocols

For our full array of protocols, please check out our website page here: https://www.molecularinstruments.com/hcr-rnafish-protocols. Listed below are several validated HCR protocols for your reference:

Images

Multiplexed protein imaging via HCR 2° IHC using unlabeled primary antibody proves, initiator-labeled secondary antibody probes and simultaneous HCR signal amplification for all targets
Multiplexed protein imaging via HCR 2° IHC using unlabeled primary antibody proves, initiator-labeled secondary antibody probes and simultaneous HCR signal amplification for all targets936×642 357 KB

Epifluorescence imaging of 4-plex protein imaging in FFPE mouse brain sections. Target proteins: TH (yellow), GFAP (green), PVALB (red), and MBP (blue). Sample: FFPE C57BL/6 mouse brain section (coronal); 5 µm thickness.

Simultaneous multiplexed protein and RNA imaging via HCR 1° IHC and HCR RNA-ISH using initiator-labeled primary antibody probes for protein targets, split-initiator DNA probes for RNA targets, and simultaneous HCR signal amplification for all targets

(A) Epifluorescence image of 4-plex protein and RNA imaging in FFPE mouse brain sections. Targets: TH (protein – green), MBP (protein – blue), Prkcd (mRNA – yellow), and Slc17a7 (mRNA – red). Sample: FFPE C57BL/6 mouse brain section (coronal); 5 µm thickness. (B) Zooms of indicated regions in A.

References

  • A. G. Ferreira, A., Sieriebriennikov, B., & Whitbeck, H. (2021). HCR RNA-FISH protocol for the whole-mount brains ofDrosophila and other insects v1. doi: 10.17504/protocols.io.bzh5p386
  • Anderson, S., Roberts, J., Zhang, J., Steele, M., Romero, C., Bosco, A., & Vetter, M. (2019). Developmental Apoptosis Promotes a Disease-Related Gene Signature and Independence from CSF1R Signaling in Retinal Microglia. Cell Reports, 27(7), 2002-2013.e5. doi: 10.1016/j.celrep.2019.04.062
  • Chen, R., Gore, F., Nguyen, Q. A., Ramakrishnan, C., Patel, S., Kim, S. H., Raffiee, M., Kim, Y. S., Hsueh, B., Krook-Magnusson, E., Soltesz, I., & Deisseroth, K. (2021). Deep brain optogenetics without intracranial surgery. Nature biotechnology, 39(2), 161–164. doi: 10.1038/s41587-020-0679-9
  • Kramer, E., Steadman, P., Epp, J., Frankland, P., & Josselyn, S. (2018). Assessing Individual Neuronal Activity Across the Intact Brain: Using Hybridization Chain Reaction (HCR) to Detect Arc mRNA Localized to the Nucleus in Volumes of Cleared Brain Tissue. Current Protocols In Neuroscience, 84(1), e49. doi: 10.1002/cpns.49
  • Kumar, V., Krolewski, D. M., Hebda-Bauer, E. K., Parsegian, A., Martin, B., Foltz, M., Akil, H., & Watson, S. J. (2021). Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues. Brain structure & function, 226(2), 481–499. doi: 10.1007/s00429-020-02194-4
  • Nicovich, P., Taormina, M., Baker, C., Nguyen, T., Thomsen, E., & Garren, E. et al. (2019). Multimodal cell type correspondence by intersectional mFISH in intact tissues. doi: 10.1101/525451
  • Park, Y., Sohn, C., Chen, R., McCue, M., Yun, D., & Drummond, G. et al. (2018). Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nature Biotechnology, 37(1), 73-83. doi: 10.1038/nbt.4281
  • Schwarzkopf, M., Liu, M., Schulte, S., Ives, R., Husain, N., Choi, H., & Pierce, N. (2021). Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization. Development, 148(22). doi: 10.1242/dev.199847
  • Shah, S., Lubeck, E., Schwarzkopf, M., He, T., Greenbaum, A., & Sohn, C. et al. (2016). Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing. Development, 143(15). doi: 10.1242/dev.138560
  • Wang, Y., Eddison, M., Fleishman, G., Weigert, M., Xu, S., & Wang, T. et al. (2021). EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell, 184(26), 6361-6377.e24. doi: 10.1016/j.cell.2021.11.024

Looking to try HCR? Use this link to register for access to a free HCR RNA-FISH Starter Kit: HCR.store/PainForum

Each HCR RNA-FISH Starter Kit will come with everything you need to conduct a 3-plex HCR RNA-FISH experiment: 3 probe sets, 3 HCR amplifiers, and a set of buffers. Each kit contains carefully selected targets that have been validated either by our expert HCR users or by the MI team.

Contact Information

Have Questions?
Post any questions or comments in this blog, and we’ll be happy to respond! If in need of technical support, please email us at support@molecularinstruments.com, and one of our scientists will be in contact with you shortly.

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If anyone here uses the HCR starter kits, let us know how your results turn out.

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From the folks who got the trial kit, any feedback? I’m curious how this is working out for people.