Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously. These properties that make HCR signal amplification well-suited for RNA-ISH appear equally favorable in the context of IHC, suggesting the approach of combining HCR signal amplification with antibody probes (Koos et al., 2015; Husain, 2016; Lin et al., 2018b). Here, we extend the benefits of one-step, quantitative, enzyme-free signal amplification from RNA-ISH to IHC, validating multiplexed, quantitative, high-resolution imaging of protein expression with high signal-to-background in highly autofluorescent samples, thus overcoming the longstanding shortcomings of IHC using CARD. Moreover, we establish a unified framework for simultaneous multiplexed, quantitative, high-resolution IHC and RNA-ISH, with one-step HCR signal amplification performed for all targets simultaneously.
Using HCR 2°IHC, protein targets are detected using unlabeled primary antibody probes that are in turn detected by secondary antibody probes labeled with one or more HCR initiators (Fig. 1D). This approach has the advantage that validation of a small library of initiator-labeled secondary antibodies (e.g. five secondaries targeting different host species) enables immediate use of large libraries of primary antibody probes (e.g. 105 commercially available primaries) without modification. On the other hand, for multiplexed experiments, each target must be detected using a primary antibody raised in a different host species to enable subsequent detection by an anti-host secondary antibody probe that triggers an orthogonal spectrally distinct HCR amplifier. Hence, depending on the available antibody probes, one may prefer HCR 1°IHC in one instance and HCR 2°IHC in another. RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context.
Epifluorescence microscopy was performed using a Leica THUNDER Imager 3D cell culture epifluorescence microscope equipped with a Leica LED8 multi-LED light source and sCMOS camera (Leica DFC9000 GTC). All epifluorescence images were acquired without THUNDER computational clearing and are displayed with instrument noise subtracted but without background subtraction. See Table S6 for details on the objective, excitation wavelengths and filters used for each experiment.
5 demonstrates HCR 1°IHC + HCR RNA-ISH (2-plex protein + 2-plex RNA) in mammalian cells and FFPE mouse brain sections using initiator-labeled primary antibody probes for protein targets, split-initiator DNA probes for RNA targets, and simultaneous HCR signal amplification for all targets. 6 demonstrates HCR 2°IHC + HCR RNA-ISH (2-plex protein + 2-plex RNA) in mammalian cells and FFPE mouse brain sections using unlabeled primary antibody probes and initiator-labeled secondary antibody probes for protein targets, split-initiator DNA probes for RNA targets, and simultaneous HCR signal amplification for all targets. HCR RNA-ISH provides automatic background suppression throughout the protocol, ensuring that reagents will not generate amplified background even if they bind non-specifically within the sample (Choi et al., 2018). During the detection stage, each RNA target is detected by a probe set comprising one or more pairs of split-initiator probes, each carrying a fraction of HCR initiator i1 (Fig. 1B).
Confocal microscopy was performed using a Zeiss LSM 800 inverted confocal microscope or a Zeiss LSM 880 with Fast Airyscan upright confocal microscope. See Table S5 for details on the microscope, objective, excitation lasers, beam splitters and emission bandpass filters used for each experiment. The aim is to dominate the game board by outmaneuvering rivals and eliminating their orbs. Achieving critical mass in these cells triggers explosive chain reactions, securing territory for the controlling player. A player can only place orbs in empty cells or in those that are already housing orbs of their color.
We have previously demonstrated that HCR RNA-ISH overcomes the historical tradeoff between RNA quantitation and anatomical context, enabling mRNA relative quantitation (qHCR imaging) with subcellular resolution within whole-mount vertebrate embryos (Trivedi et al., 2018; Choi et al., 2018). Here, we demonstrate that HCR IHC enables analogous subcellular quantitation of proteins in an anatomical context. To test protein relative quantitation, we first redundantly detected a target protein using two primary antibody probes that bind different epitopes on the same protein and trigger different spectrally distinct HCR amplifiers (Fig. 4A; top), yielding a two-channel image (Fig. 4B; top). If HCR signal scales approximately linearly with the number of target proteins per voxel, a two-channel Chain Reaction 2.0 bot scatter plot of normalized voxel intensities will yield a tight linear distribution with approximately zero intercept (Trivedi et al., 2018). On the other hand, observing a tight linear distribution with approximately zero intercept (Fig. 4C; top), we conclude that the HCR signal scales approximately linearly with the number of target proteins per imaging voxel, after first ruling out potential systematic crowding effects that could permit pairwise voxel intensities to slide undetected along a line (Fig. S24). Using one initiator-labeled primary antibody probe per channel, we observe high accuracy (linearity with zero intercept) and precision (scatter around the line) for subcellular 2×2 µm voxels within 5 µm FFPE mouse brain sections using epifluorescence microscopy.
Both methods achieve high signal-to-background for 3-plex protein imaging in mammalian cells and for 4-plex protein imaging in FFPE mouse brain sections. It is important for biologists, drug developers and pathologists to have the flexibility to image proteins and RNAs simultaneously so as to enable interrogation of both levels of gene expression in the same specimen. Here, we demonstrate that HCR 1°IHC and HCR 2°IHC are both compatible with HCR RNA-ISH, enabling multiplexed quantitative protein and RNA imaging with high signal-to-background.
Nonetheless, during the amplification stage, kinetically trapped HCR hairpins provide automatic background suppression for protein targets just as they do for RNA targets, ensuring that any hairpins that bind non-specifically in the sample do not trigger growth of an HCR amplification polymer. For experiments using HCR IHC + HCR RNA-ISH to image protein and RNA targets simultaneously, RNA targets enjoy automatic background suppression throughout the protocol, whereas protein targets rely on selective antibody binding to suppress background during the detection stage, combined with automatic background suppression during the amplification stage. To image RNA expression, targets are detected by nucleic acid probes that trigger isothermal enzyme-free chain reactions in which fluorophore-labeled HCR hairpins self-assemble into tethered fluorescent amplification polymers (Fig. 1B). Orthogonal HCR amplifiers operate independently within the sample so the experimental timeline for multiplexed experiments is independent of the number of target RNAs (Choi et al., 2010, 2014). The amplified HCR signal scales approximately linearly with the number of target molecules (Fig. 1E), enabling accurate and precise RNA relative quantitation with subcellular resolution in the anatomical context of whole-mount vertebrate embryos (Trivedi et al., 2018; Choi et al., 2018). Amplification polymers remain tethered to their initiating probes, enabling imaging of RNA expression with subcellular or single-molecule resolution as desired (Choi et al., 2014, 2016, 2018; Shah et al., 2016).
This redundant detection experiment provides a conservative characterization of quantitative performance as there is the risk that two antibody probes may interfere with each other to some extent when attempting to bind different epitopes on the same target protein. As a further test of quantitative imaging characteristics, we detected a protein target with unlabeled primary antibody probes that are subsequently detected by two batches of secondary antibody probes that trigger different spectrally distinct HCR amplifiers (Fig. 4A; bottom). This experiment is testing the accuracy and precision of the secondary antibody probes and HCR signal amplification, but not that of the primary antibody probes. In FFPE human breast tissue sections using confocal microscopy (Fig. 4B; bottom), a two-channel scatter plot of voxel intensities for subcellular 2.0×2.0×2.5 µm voxels again reveals a tight linear distribution with approximately zero intercept (Fig. 4C; bottom).
A large number of rivals have returned from the previous game, and there are new rival types such as the Lovers; female drivers who send the player mail upon beating them. Beating rivals can also unlock musical samples for the player to create their own music in the game’s “Sound Editor” mode. What stands out in this strategy game is the tension and tactical depth as players work to ensure they are not the first to lose all their orbs and get eliminated from the intense competition. It offers an immersive experience with high-definition modes for larger screens and a regular mode suitable for all types of devices.
Personalization is also a priority; it allows customization of the appearance and sound of the orbs, as well as the option to toggle tactile feedback, which augments the tactile element of the gameplay experience. Chain Reaction 2023, like all THW games covering a variety of genres, is designed to be played solo, cooperatively – all players on the same side – or competitively – head-to-head. Chain Reaction 2023 is a set of man-to-man combat rules that can be played in a variety of periods and with any figures you may already have.
For a given probe pair, probes that hybridize specifically to their adjacent binding sites on the target RNA colocalize full initiator i1, enabling cooperative initiation of HCR signal amplification. Meanwhile, any individual probes that bind non-specifically in the sample do not colocalize full initiator i1, do not trigger HCR and thus suppress generation of amplified background. During the amplification stage, automatic background suppression is inherent to HCR hairpins because polymerization is conditional on the presence of the initiator i1; individual h1 or chainreaction20.co.uk h2 hairpins that bind non-specifically in the sample do not trigger formation of an amplification polymer. For HCR IHC, during the detection stage, each target protein is detected using primary or secondary antibody probes carrying one or more full i1 initiators (Fig. 1C,D). Hence, if an antibody probe binds non-specifically in the sample, initiator i1 will nonetheless trigger HCR, generating amplified background. As a result, it is important to use antibody probes that are highly selective for their targets, and to wash unused antibody probes from the sample.
In addition to the previously introduced CA (Cornering Artist) and SA (Spirit Attack) modes, this sequel also features new gameplay modes such as FL/LF (First and Last/Last and First) battle modes, TAC (Time Attack Collision) or CAT (Cornering Artist Target) battles. With simple yet deep mechanics and extensive customization options, the game promises a dynamic experience that will test players’ strategic abilities. Whether on a tablet or smartphone, users can prepare to engage in an electrifying battle to be the last one standing on the digital board.
Based on these two studies, we conclude that qHCR imaging enables accurate and precise relative quantitation of protein targets in an anatomical context with subcellular resolution, just as it does for mRNA targets (Trivedi et al., 2018; Choi et al., 2018). 2 demonstrates multiplexed protein imaging via HCR 1°IHC using initiator-labeled primary antibody probes. 3 demonstrates multiplexed protein imaging via HCR 2°IHC using unlabeled primary antibody probes and initiator-labeled secondary antibody probes.