Single-plex chromogenic HCR staining

As illustrated in the experimental schematic in Fig. 1, probe pairs hybridize to the target mRNA sequence, thereby forming HCR initiators on the mRNA. HCR was subsequently performed using hairpin DNA conjugated with haptens, such as biotin, resulting in the localized accumulation of these haptens around the mRNA. Following the HCR reaction, POD- or AP-conjugated streptavidin was applied to the biotin-labeled sections, and POD- or AP-conjugated anti-hapten antibody was applied to the DIG or fluorescein-labeled sections. Finally, color development by POD and AP substrate enabled the visualization of mRNA in the tissue.

First, we investigated how different combinations of haptens and enzymes affect staining behavior on floating sections of mouse brains. As shown in Fig. 2, Penk mRNA was successfully detected in the mouse brain tissue sections using chromogenic in situ HCR, regardless of whether the hairpin DNA was labeled with DIG, fluorescein, or biotin. These signals were not observed when only probes and antibodies were reacted without hairpin DNA, confirming the specificity of the antibodies (Fig. 2g–i and s–u). All labeling strategies yielded a similar and specific distribution of Penk mRNA in the CP and piriform cortex (PIR), consistent with the expression pattern observed in the fluorescent in situ HCR staining (Fig. 2v,w) and previous reports (Turchan et al. 1997; Lobo et al. 2006; Labouesse et al. 2018). Such Penk expression distributions were not observed in either the no-probe control, the unpaired-probe control (targeting Penk in the first half and Esr1 in the second half probes), or RNase-pretreated sections (Fig. 2j–l), suggesting that the observed chromogenic staining reflects specific Penk expression. However, the signal intensity varied among the haptens. DIG and fluorescein hairpin DNA produced comparable signals, while the biotin–streptavidin combination produced the strongest signals when detected with either POD (Fig. 2a–f) or AP (Fig. 2m–r). In addition, streptavidin-based detection provided a slightly higher signal-to-noise ratio than antibody-based detection, regardless of the labeling enzyme (Fig. 2x). Such differences in staining intensity among haptens are likely attributable to affinity of tags and variations in immunohistochemical detection methods. Although the avidin–biotin complex (ABC) method is another approach for detecting biotin conjugate, the signal tends to be much less intense when using the ABC method than the streptavidin–biotin method in chromogenic HCR, consistent with a previous report (McQuaid and Allan 1992). POD-polymer-conjugated streptavidin was sometimes used in immunohistochemical detection to enhance signal intensity, but it shows little difference in signal intensity compared with streptavidin conjugated with POD monomer (data not shown). These observations suggest that detection sensitivity is influenced not only by the differences in affinity for the haptens but also by factors such as tissue permeability and molecular size of the antibodies or streptavidin used for readout. Given its high sensitivity, the streptavidin–biotin staining method is often the preferred choice in chromogenic HCR staining. Nonetheless, in cases where endogenous biotin leads to unavoidable background, alternative tags such as DIG or fluorescein may offer more reliable detection.

Next, we attempted to visualize genes with low expression levels using chromogenic HCR staining in the mouse brain (Fig. 3). Oxtr mRNA was stained using biotinylated hairpin DNA combined with POD-conjugated streptavidin. Numerous granular brown signals were observed in the hippocampal CA3 region (Fig. 3a), consistent with the distribution observed in fluorescent HCR staining of Oxtr mRNA (Fig. 3c). However, no signal was observed with the unpaired probe targeting Oxtr and Drd1. The distribution of Oxtr localized in the CA3 region is further supported by the observation from OXTR-VENUS mice (Lin et al. 2017). There was no significant difference in puncta counts per cell between chromogenic and fluorescence staining in the CA3 region (Fig. 3d). In addition, we examined Oxtr expression in the lateral septum (LS), medial nucleus of the hypothalamus (MN), anterior cingulate cortex (ACC), and endopiriform nucleus, where Oxtr expression has been previously reported (Yoshimura et al. 1996; Ostrowski 1998; Tsuneoka et al. 2013; Menon et al. 2018; Sharma et al. 2019; Tsuneoka and Funato 2020; Hidema et al. 2023; Li et al. 2024), and observed specific expression patterns in these regions (Fig. 3e–h). We also examined sections from the mouse medial preoptic area (MPOA), where Oxtr expression is known to increase postpartum (Meddle et al. 2007). In the MPOA, mothers showed higher numbers of Oxtr-positive cells, higher total Oxtr puncta counts, and higher puncta counts per cell than virgin mice (Fig. 3i–m). Fluorescence in situ HCR is a highly quantitative ISH method with single-copy detection sensitivity. The comparison with fluorescence staining in the CA3 region suggests that chromogenic in situ HCR has sensitivity and resolution comparable to fluorescence, with minimal nonspecific signal. Furthermore, the comparison between virgin and maternal mice in the MPOA suggests that the high quantitative performance is also achievable with chromogenic HCR.

Fig. 3The alternative text for this image may have been generated using AI.

Representative images of chromogenic HCR staining for Oxtr and Drd2 mRNA in the mouse brain. a, c Oxtr mRNA in the CA3 region of the hippocampus. a HCR was performed using biotin-conjugated hairpin DNA, bound by POD-conjugated streptavidin, and visualized by DAB substrate. b Unpaired probe control of Oxtr and Drd1. c HCR was performed using ATTO550-conjugated hairpin DNA. d Comparison of puncta counts/cell between chromogenic and fluorescent HCR staining of Oxtr. e–h Oxtr Expression in the lateral septum (LS) (e), magnocellular nucleus (MN) (f), anterior cingulate cortex (ACC), layer 1 and 2/3 (g), and endopiriform nucleus (h). i–m Oxtr Expression difference between virgin female (n = 4) and mother (n = 5) in the medial preoptic area (MPOA). i Representative expression in virgin MPOA. j Representative expression in mother MPOA. k Difference in Oxtr-positive cell number. l Difference in total Oxtr-positive puncta. m Difference in Oxtr puncta for individual cells (virgin: 243 cells; mother: 658 cells). n, p Drd2 mRNA in the dentate gyrus (DG). n HCR was performed using DIG-conjugated hairpin DNA, bound by AP-conjugated anti-DIG antibody, and visualized by VectorRed substrate. o Unpaired probe control of Drd2 and Lrp. p HCR was performed using Cy5-conjugated hairpin DNA. Arrowheads indicate the Drd2 mRNA-positive cells. q Comparison of puncta counts/cell between chromogenic and fluorescent HCR staining of Drd2. r–u Drd2 expression in caudate putamen (CP) (r), paraventricular thalamic nucleus (PVT) (s), endopiriform nucleus (t), and zona incerta (ZI) (u). Bars represent median values (d, m, q) and means (k, l). Scale bars: 25 μm (i–j), 50 μm (others). DGp dentate gyrus, polymorphic layer, DGg dentate gyrus, granule cell layer, fr fasciculus retroflexus

A combination of DIG-conjugated hairpins and AP-conjugated anti-DIG antibodies successfully visualized Drd2 mRNA, and neurons containing many pink–red granules were observed in the dentate gyrus polymorphic layer (DGp), while signal was not observed with the unpaired probe targeting Drd2 and Lrp (Fig. 3n,o). Similarly, neurons containing many fluorescent granules were observed in the DGp in the fluorescently stained sections using SaraFluor488-labeled hairpins (Fig. 3p), and such a distribution pattern was also observed in the previous report (Etter and Krezel 2014). Drd2-positive puncta counts per cell were not substantially different between chromogenic and fluorescence HCR (Fig. 3q). Drd2 expression was observed in the CP, paraventricular thalamic nucleus (PVT), endopiriform nucleus, and zona incerta (ZI) (Fig. 3r–u). These specific expression patterns are consistent with previous reports (Clark et al. 2017; Wang et al. 2019; Khlghatyan et al. 2019; Gao et al. 2020), suggesting that Drd2 expression is faithfully visualized in chromogenic HCR.

To assess the availability of chromogenic HCR in slide-mounted sections in addition to the floating sections, periostin and Esr1 mRNA was stained by chromogenic HCR in smooth muscle of the stomach and epididymal ducts, respectively (Fig. 4). Periostin mRNA was visualized as brown granules using biotinylated hairpin DNA and streptavidin POD (Fig. 4a), revealing that a subset of non-spindle-shaped cells within the smooth muscle tissue were positive. These cell-type-specific signals of periostin mRNA were not observed in the no-probe control (Fig. 4b). The granule density in the cells and the morphology of positive cells were similar when visualized by fluorescent direct staining using Atto550-conjugated hairpin DNA (Fig. 4c,d). Esr1 mRNA, which is known to be weakly expressed in the epididymal tubular epithelium (Zhou et al. 2002; Yamashita 2004), was detected in the epithelium of epididymal ducts using DIG-conjugated hairpin DNA and AP-conjugated anti-DIG antibodies. Many pink–red granules were observed in the ductal epithelium, whereas no signal was detected in the sperm within the lumen or in the no-probe control (Fig. 4e,f). This specific pattern was similarly observed with fluorescent direct staining using Cy5-conjugated hairpin DNA (Fig. 4g,h).

Fig. 4The alternative text for this image may have been generated using AI.

Representative images of chromogenic HCR staining for Periostin and Esr1 mRNA in slide-mounted sections. a, c Periostin mRNA in the smooth muscle of the stomach. a HCR was performed using biotin-conjugated hairpin DNA, bound by POD-conjugated streptavidin, and visualized by DAB substrate. b No-probe control. c HCR was performed using ATTO550-conjugated hairpin DNA. Arrowheads indicate the Periostin mRNA-positive cells. d Comparison of puncta counts/cell between chromogenic and fluorescent HCR staining of Periostin. e, g Esr1 mRNA in the epithelium of the epididymal ducts. e HCR was performed using DIG-conjugated hairpin DNA, bound by AP-conjugated anti-DIG antibody, and visualized by VectorRed substrate. f No-probe control. g HCR was performed using Cy5-conjugated hairpin DNA. h Comparison of puncta counts/cell between chromogenic and fluorescent HCR staining of Esr1. Scale bars: 50 μm

These results showed that granular signals similar to those observed with fluorescent HCR staining were also detected by chromogenic bright-field staining in both floating and mounted tissue sections. While free-floating sections allow antibody penetration from both sides, mounted sections permit access only from one side, making it difficult to achieve identical staining conditions. Nevertheless, by optimizing antibody concentration and incubation time (Table 1), we were able to obtain comparable staining intensity across free-floating and mounted sections. These observations suggest that, using the protocol proposed in this study, chromogenic detection of HCR signals can achieve sensitivity comparable to that of fluorescence-based methods. Although indirect chromogenic visualization involves more procedural steps than direct fluorescence staining, it is particularly useful for detecting mRNA in tissues with strong autofluorescence, where fluorescent signals may be obscured, and for samples intended for long-term preservation.

POD and AP each have their own advantages and limitations. POD offers superior signal localization and rapid color development, whereas AP allows for stronger signal generation through prolonged incubation in substrate-containing buffer. Although there are differences in signal contrast, the fact that similar staining results were obtained with substrates for both POD and AP indicates that the choice of enzyme and substrate combinations can be flexibly adapted to suit the target tissue and gene.

Given that fluorescent in situ HCR has been successfully applied to formalin‑fixed paraffin‑embedded (FFPE) tissues without additional procedural modifications (Wong et al. 2023; Kuboe et al. 2025), and that fluorescent and chromogenic HCR share the same underlying hybridization and amplification mechanism, we anticipate that the chromogenic HCR protocol can be directly extended to FFPE specimens. Although the present study was performed on cryosections, preliminary experiments indicate that this chromogenic HCR protocol is applicable to FFPE tissues. Future work will include quantitative validation of chromogenic HCR performance in clinical FFPE material.

Duplex chromogenic HCR staining

To evaluate feasibility of simultaneous visualization of two target mRNAs, probe hybridization and HCR reactions for two targets were performed simultaneously, followed by sequential hapten detection and chromogenic staining using the same procedure as for the single staining (Fig. 5). In mouse brain, Penk mRNA is specifically expressed in the subset of neurons within the CP, where the somata exhibited intense red staining using AP and VectorRed substrate. Vglut1 mRNA expression is specific to the cerebral cortex, with cells showing intense red and brown signals upon POD/DAB and AP/VectorRed staining, respectively (Fig. 5a,b and i,j). Drd1 is expressed in a distinct neuronal subset from Penk mRNA-positive neurons in the CP (Gokce et al. 2016), and brown-stained granules of Drd1 by POD/DAB were also observed in the intercalated cell nucleus (ICN; Fig. 5j). These red and brown signals were separable and maintained specificity in expression patterns, even under conditions of duplex staining, and the direct fluorescent staining using fluorophore-conjugated hairpin DNA showed comparable distributions (Fig. 5c,g,k). Such specific staining was not observed in the no-probe control (Fig. 5d,h,l). Importantly, the comparable signal intensity observed under dual-staining conditions indicates that chromogenic HCR retains its sensitivity and can be applied with the same reliability as in single-staining setups. This expands its utility in multiplexed histological analyses without compromising detection performance.

Fig. 5The alternative text for this image may have been generated using AI.

Representative images of duplex chromogenic HCR staining and corresponding fluorescent HCR staining. a–l Duplex staining in the mouse brain. a, b Penk and Vglut1 mRNA was detected using a combination of DIG/AP-labeled anti-DIG antibody with VectorRed and fluorescein/POD-labeled anti-fluorescein antibody with DAB, respectively. c Penk and Vglut1 fluorescent HCR staining of the same fields. d No-probe control. e, f Penk and Drd1 mRNA was detected using a combination of DIG/AP-labeled anti-DIG antibody with VectorRed and biotin/POD-labeled streptavidin with DAB, respectively. g Penk and Drd1 fluorescent HCR staining of the same fields. h No-probe control. i, j Vglut1 and Drd1 mRNA was detected using a combination of fluorescein/AP-labeled anti-fluorescein antibody with VectorRed and biotin/POD-labeled streptavidin antibody with DAB, respectively. k Vglut1 and Drd1 fluorescent HCR staining of the same fields. h No-probe control. Panels b, f, j show magnified views of the rectangle regions indicated in (a, e, i). m Cyp2e1 and albumin mRNA in the mouse liver was detected using a combination of DIG/AP-labeled anti-DIG antibody with BCIP/NBT and biotin/POD-labeled streptavidin with DAB, respectively. n Fluorescent HCR staining of the same region as (m). o No-probe control. p Lrp2 and Umod mRNA in the mouse kidney was detected using a combination of fluorescein/AP-labeled anti-fluorescein antibody with BCIP/NBT and biotin/POD-labeled streptavidin with DAB, respectively. s Fluorescent HCR staining of the same region as (p). q Magnified view of the rectangle region indicated in (p). r Renal cortex image from the sections stained using VectorRed as the substrate for AP instead of BCIP/NBT. t, u No-probe control. Scale bars: 500 μm (a, c, d, e, g, h, i, k, l, p, s), 100 μm (b, f, j), 200 μm (m–o), 25 μm (q, r, t, u). CP caudate putamen, CTX cortex, ICN intercalated nucleus, Pv portal vein, Cv central vein, C cortex, M medulla, PT proximal tubule, DT distal tubule

In the liver, there is a gene expression gradient from the portal to central venous regions, and albumin and Cyp2e1 mRNA is complementarily expressed along this axis (Ghafoory et al. 2013; Hu et al. 2022). Albumin mRNA was labeled with biotinylated hairpin DNA and visualized chromogenically using streptavidin POD and DAB, showing strong expression in the hepatocyte around the portal vein (Fig. 5m). In parallel, Cyp2e1 mRNA was labeled with DIG-conjugated hairpin DNA and visualized using AP-conjugated anti-DIG antibody and BCIP/NBT substrate. Cyp2e1 mRNA was strongly and specifically expressed in hepatocytes around the central vein. These trends were also confirmed in the fluorescent direct staining (Fig. 5n), and no-probe control experiments showed endogenous biotin or enzyme did not produce the region-specific staining (Fig. 5o). These findings suggest that dual staining can be successfully performed not only in floating sections but also in mounted sections, with chromogenic HCR yielding results comparable to fluorescent staining. However, in cases involving highly abundant mRNA such as albumin or Cyp2e1 in the liver, the nature of bright-field detection makes it challenging to determine whether two transcripts are colocalized within the same cell. This limitation warrants caution and indicates that such applications may not be practical. Therefore, to reliably assess colocalization in dual staining setups, careful selection and optimization of enzyme–substrate combinations are essential. It should be noted that dark counterstaining may interfere with the visualization of mRNA signals, and thus requires careful optimization to avoid masking target-specific staining.

In the kidney, Lrp2 and Umod were expressed as markers of proximal and distal tubules, respectively (Tokonami et al. 2018; Rudman-Melnick et al. 2020; Aceves et al. 2022). Lrp2 mRNA was labeled with fluorescein-conjugated hairpin DNA and visualized using AP-conjugated anti-fluorescein antibody with either BCIP/NBT or VectorRed as the AP substrate. Umod mRNA was labeled with DIG-conjugated hairpin DNA and visualized using POD-conjugated anti-DIG antibody and DAB. The kidney contains abundant endogenous biotin, and our preliminary no-probe control experiments showed strong background signals attributable to endogenous biotin. Therefore, we avoided biotin–streptavidin-based detection in this tissue and instead used DIG- and fluorescein‑based detection systems. As expected, the expression of Lrp2 and Umod showed exclusive distribution. Lrp2 mRNA was confined to tubes in the renal cortex, while Umod mRNA was observed both in the cortex and medulla, suggesting specific staining of the proximal and distal tubules, respectively (Fig. 5p,q,r). These staining patterns were consistent with those obtained by fluorescent direct staining (Fig. 5s). To compare two major AP substrates for the chromogenic visualization, epithelial cells in the proximal tubules were stained blue and red when BCIP/NBT and VectorRed were used, respectively. No signal was detected in either negative control using BCIP/NBT or VectorRed as the chromogenic substrate (Fig. 5t,u). The overall staining distribution was similar between these two AP substrates used for Lrp2 mRNA detection, and the signals were biased toward the luminal side and located beneath the apical surface of the proximal tubules. The staining characteristics varied depending on the substrate used. BCIP/NBT produced diffuse cytoplasmic staining, whereas VectorRed yielded concentrated, granular signals (Fig. 5q,r). In addition to their distinct optical properties, which directly affect microscopic visualization, the precision of pigment localization and the stability of the reaction product are also critical factors influencing the overall staining outcome. BCIP/NBT, which produces a blue-to-purple precipitate, offers excellent contrast and visibility against the brown coloration of DAB. However, owing to its tendency to diffuse, it may be less suitable for precise localization of mRNA signals. In bright-field microscopy, spatial resolution is inherently limited by light diffraction and the spread of chromogenic precipitates, resulting in lower resolution than fluorescence-based detection. Bright-field imaging also lacks the optical sectioning capability of confocal microscopy, making it difficult to resolve signals from overlapping cells along the z-axis. Consequently, signals from adjacent cells or closely spaced subcellular compartments can appear to overlap, even when mRNA is expressed in distinct cells or regions. For these reasons, true colocalization at the single-cell level cannot be reliably assessed by bright-field chromogenic duplex staining alone. Although duplex chromogenic HCR can reveal regional co-expression patterns, it does not provide sufficient spatial resolution to unequivocally determine co-expression within individual cells. In tissues with high cell density or overlapping signals, apparent color mixing may reflect signal overlap from adjacent cells rather than true co-expression. However, when a brightly stained, highly expressed cellular marker is combined with a punctate mRNA signal stained in a darker color, colocalization can sometimes be inferred at the single-cell level, provided the signals are clearly distinct and non-overlapping. For rigorous assessment of single-cell co-expression, complementary approaches such as fluorescent duplex HCR or single-cell reverse transcription polymerase chain reaction (PCR) should be considered.

Next, Esr1 mRNA and its encoded protein, estrogen receptor alpha (ERα), were visualized in the mouse hypothalamus using a combination of in situ HCR and immunostaining (Fig. 6). In mouse hypothalamus, Esr1 mRNA is known to be highly expressed in the arcuate nucleus (ARC), ventrolateral part of the ventromedial hypothalamus (VMHvl), and anterior hypothalamic nucleus pars posterior (AHNp) (Simerly et al. 1990; Shughrue et al. 1997; Mitra et al. 2003). Using chromogenic and fluorescent staining, we confirmed the presence of both granule-like signals of Esr1 mRNA and nuclear ERα immunoreactivity in the ARC, VMHvl, and AHN (Fig. 6a–c). The number of Esr1-positive granules per cell was high in the ARC and VMHvl, whereas the AHNp exhibited a lower density of Esr1 positive granules per cell (Fig. 6d–i). Similarly, the ERα immunoreactivity was more intense in the ARCs and VMHvl and weaker in the AHNp (Fig. 6d–i). However, when the positive cells were observed separately, the number of Esr1 mRNA-positive granules and intensity of ERα immunostaining did not always correlate. In some cells, a high number of Esr1-positive granules was accompanied by weak ERα immunoreactivity. These patterns were consistently observed in both chromogenic and fluorescent staining (Fig. 6d–i). These staining results reinforce a key advantage of in situ HCR using short hairpins, namely its compatibility with costaining by immunohistochemistry (Tsuneoka and Funato 2020). In the ARC and VMH, the number of Esr1 mRNA puncta per ERα-positive cell did not differ between chromogenic and fluorescence HCR, whereas a significant difference was observed in the AHNp (Fig. 6j). The difference in the AHNp may be due to regional variations in background signal, particularly in fluorescence observations, where part of the signals may have fallen below the detection threshold. Chromogenic HCR staining delivered sensitivity and spatial resolution comparable to fluorescent HCR when combined with IHC, supporting its use as an effective alternative for experiments requiring simultaneous detection of IHC. The combination of nuclear IHC markers and cytoplasmic HCR granules is especially useful for assessing intracellular colocalization because the nuclear signal supplies a clear cellular landmark, while HCR reveals transcript distribution, reducing ambiguity when assigning signals to the same cell. In many ISH methods, treatments such as pepsin or proteinase K digestion are required to enhance probe penetration and ensure sensitivity (Tsuneoka and Funato 2020). However, these treatments often markedly reduce the sensitivity of subsequent immunostaining and add complexity by increasing the number of procedural steps. In contrast, the in situ HCR method employing short hairpins enables chromogenic staining through a streamlined protocol and offers the advantage of allowing immunostaining to be performed sequentially.

Fig. 6The alternative text for this image may have been generated using AI.

Representative images of in situ HCR staining for Esr1 mRNA and immunostaining for ERα protein in the mouse hypothalamus. a–c Wide-field view of HCR staining sections. a, d–f Esr1 mRNA was visualized using a combination of biotin-conjugated hairpin DNA, POD-conjugated streptavidin, and DAB substrate. b, c, g–i Fluorescent staining of Esr1 mRNA and ERα protein. d–i Magnified view of regions marked by rectangles (a–c). j Comparison of puncta counts/ERα-positive cell between chromogenic and fluorescent HCR staining of Esr1. Scale bars: 200 μm (a–c) or 25 μm (d–i). AHNp anterior hypothalamic nucleus, posterior part, ARC arcuate nucleus, VMHvl ventromedial hypothalamic nucleus, ventrolateral part

The relationship between Esr1 mRNA expression and ERα protein abundance is notable. The regional trends of expression level of mRNA were similar to those of protein. This parallel pattern supports region-specific regulation of Esr1 transcription and indicates that the in situ HCR accurately identifies the subpopulation of Esr1 expressing cells in the hypothalamus. However, discordance between mRNA and protein levels in individual cells suggests that either transcription, translation, or stability of mRNA and protein differs cell by cell, or that the peaks of mRNA and protein expression are temporally offset (Liu et al. 2016; Genshaft et al. 2016).

Our chromogenic HCR protocol offers several practical advantages compared with established chromogenic ISH platforms such as RNAscope, a branched DNA-based assay. First, regarding workflow complexity, chromogenic HCR does not require protease K digestion under the conditions used here, whereas RNAscope-type assays typically rely on protease treatment. Second, regarding cost, the reagent cost per sample for chromogenic HCR is substantially lower than that of commercial RNAscope assays; in our experience in Japan, the cost is less than one-tenth of that for RNAscope. Third, with respect to instrumentation, chromogenic HCR can be performed using standard laboratory equipment without specialized devices. In terms of multiplexing, chromogenic HCR is limited to two colors in bright-field microscopy owing to the availability of chromogenic substrates and the difficulty of distinguishing more than two colors. Advantages of chromogenic HCR include its simple workflow, low cost, no requirement for specialized equipment, and flexibility in probe design. Limitations include restricted multiplexing capacity and the need to optimize antibody/streptavidin-based detection conditions for each tissue type, whereas RNAscope-type assays do not rely on antibodies for signal detection. Despite these limitations, chromogenic HCR provides a cost-effective and accessible alternative to commercial branched DNA platforms, particularly for research laboratories seeking flexibility in probe design and simplified workflows.

In conclusion, we confirmed, across various specimen types, that the chromogenic staining method we developed using in situ HCR achieves detection sensitivity comparable to that of in situ HCR fluorescence staining. Importantly, in situ HCR allows staining to be performed with the same protocol across multiple platforms, and even in bright-field chromogenic staining applications, the HCR reaction can be carried out using the identical protocol. Since differences in haptens, detection systems, and chromogenic substrates can substantially affect staining behavior, optimization of these components will be necessary. Nevertheless, this approach enables double staining without compromising the compatibility with immunostaining, thereby expanding the options for specimens in which fluorescence staining is difficult to apply.