whole-mount in situ hybridization

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Multi probe in situ hybridization to whole mount Arabidopsis seedlings

Multi probe in situ hybridization to whole mount Arabidopsis seedlings

On this basis, ISH is an essential technique to investigate gene expression at a cellular level. Methods for ISH, first developed in the 1980s, used radioactively labeled antisense RNA probes to detect expression of genes on histological sections prepared from wax embedded tissues and processed according to autoradio- graphic techniques (Harrison et al.1974). Improvements that led to safer and more accurate assays involved: i) the introduction of hapten-labeled probes that allowed the use of immunohistochemi- cal procedures for probe detection; ii) the use of fluorophore- labeled antibodies for the detection of nucleic acids and iii) the direct (i.e. without antibodies) hybridization of fluorescently la- beled nucleic acids which resolved the problems related to antibody detection through enzymatic assay per se (Bauman et al. 1980). The ISH methodologies associated with it have under- gone continuous refinement (Levsky and Singer 2003). ISH performed on sections has been widely used in many model organisms, but it is time-consuming and requires much expertise. Since the 1990s, whole mount in situ technology has elimi- nated the need for embedding procedures and has made the analysis of gene expression patterns rapid (Hejátko et al. 2006; Piette et al. 2008; Traas 2008; Vize et al. 2009). Originally, the whole mount in situ technology was introduced and optimized for transcript localization in animals (Kosman et al. 2004). In plants, the challenge was to overcome problems imposed by the cell wall, which can limit efficient penetration of the probe and hence hamper the hybridization outcome. Recently, whole mount in situ hybridization methods were optimized and work effectively in plants, using only one digoxigenin-labeled probe for a single whole mount (Hejátko et al. 2006; Traas 2008). However, there is a need for the simultaneous visualization of transcripts of several genes to discern whether they act in the same or different domains or tissue during plant development.
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Original Article An optimized protocol for whole mount in situ hybridization of mouse brain

Original Article An optimized protocol for whole mount in situ hybridization of mouse brain

Abstract: In this study, we describe an optimized protocol of whole mount in situ hybridization (WMISH) which is a preferred method for transcript distributions detection in whole embryos, tissues and organs, for the detection of c-fos mRNA in fresh frozen brain tissue. Therefore, critical steps of WMISH were optimized including proteinase K digestion time, composition of hybridization buffer as well as durations of incubation and washing steps. The ex- pression of c-fos mRNA and protein was detected in mouse brains with the modified WMISH protocol. As a result, the expression patterns of c-fos mRNA and protein in mouse brains were successfully detected with great specificity and low background signal.
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Alpha tubulin marker gene of neural territory of sea urchin embryos detected by whole mount in situ hybridization

Alpha tubulin marker gene of neural territory of sea urchin embryos detected by whole mount in situ hybridization

Int J Dev lI;o! 39 477 483 (1995) 477 Q,iginal Article a Tubulin marker gene of neural territory of sea urchin embryos detected by whole mount in situ hybridization FABRIZIO GIANGUZZA', CATERINA CASAN[.]

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Whole mount in situ hybridization reveals the expression of the Xl Fli gene in several lineages of migrating cells in Xenopus embryos

Whole mount in situ hybridization reveals the expression of the Xl Fli gene in several lineages of migrating cells in Xenopus embryos

Int J De, lIiol 39 909 919 (1995) 909 Original Article Whole mount in situ hybridization reveals the expression of the XI Fli gene in several lineages of migrating cells in Xenopus embryos DENISE MEYE[.]

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Patterns of gene expression in schistosomes: localization by whole mount in situ hybridization

Patterns of gene expression in schistosomes: localization by whole mount in situ hybridization

As a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mount in situ hybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.
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Localization of a nervous system specific class II beta tubulin gene in Xenopus laevis embryos by whole mount in situ hybridization

Localization of a nervous system specific class II beta tubulin gene in Xenopus laevis embryos by whole mount in situ hybridization

Int I De, Riol J5 399 405 (l tJ(1) 399 Original Article Localization of a nervous system specific class II B tubulin gene in Xenopus laevis embryos by whole mount in situ hybridization RALF OSCHWALD',[.]

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Postimplantation mouse embryos cultured in vitro  Assessment with whole mount immunostaining and in situ hybridization

Postimplantation mouse embryos cultured in vitro Assessment with whole mount immunostaining and in situ hybridization

Tn! J, Dey, BioI ~1 365 3N (1997) 365 Postimplantation mouse embryos cultured in vitro Assessment with whole mount immunostaining and in situ hybridization GENEVIEVE VAN MAELE FABRY, FREDERIC CLOTMAN,[.]

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Mapping Neurotransmitter Identity in the Whole-Mount Drosophila Brain Using Multiplex High-Throughput Fluorescence in Situ Hybridization

Mapping Neurotransmitter Identity in the Whole-Mount Drosophila Brain Using Multiplex High-Throughput Fluorescence in Situ Hybridization

coverslips. They were returned to 4° 100% ethanol for stor- age until beginning the main FISH protocol. One to four coverslips were moved between jars (W900180-6; Wheaton, Millville, NJ) containing 10 ml of solution for most processing steps. Multiple jars were processed in parallel when needed. Hybridization was performed in custom plexiglass chambers modified from https://hhmi.flintbox.com/public/project/ 26606/ and related designs [Figure 3A, Figure S5A, and Wu et al. (2016)]. The 22 3 22-mm coverslip is held above the bottom of the chamber by 0.5 3 5.5 3 22-mm spacers on each side, leaving an 0.5 3 11 3 22-mm space for the samples and 150–180 ml of hybridization solution. A hole at the top allows for overflow, and access for coverslip addi- tion and removal. The 20-hr hybridization reaction was car- ried out with the chambers inside a humidified polypropylene container (2249-6; Ted Pella). FISH probes were labeled with one of the following fluorophores: Cy3 (GE PA13101), Cy5 (GE PA15101), Quasar 570 (LGC Biosearch Technolo- gies), CF594 (92132; Biotium), Alexa Fluor 594 (AF594) (A20004; Thermo Fisher Scientific), DyLight550 (DL550) (62262; Thermo Fisher Scientific), or CAL Fluor 610 Red (LGC Biosearch Technologies). Please see Supplemental Ma- terial for step-by-step coverslip FISH protocol. For serotonin immunostaining with FISH, the tissues were first exposed to 1:50 mouse anti-serotonin (MS-1431-S0; Thermo Fisher Sci- entific) overnight at 4°. After washing the primary antibody, brain tissues were prepared for FISH. Next, 1:400 AF568 goat anti-mouse secondary (A-11031; Thermo Fisher Scientific) was added in the second step of hybridization and incubated along with FISH probes. After the series of wash steps de- scribed in the FISH protocol, the tissues were fixed and mounted with distyrene, plasticizer, and xylene (DPX).
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Rainbow trout glucose transporter (OnmyGLUT1): functional assessment in Xenopus laevis oocytes and expression in fish embryos

Rainbow trout glucose transporter (OnmyGLUT1): functional assessment in Xenopus laevis oocytes and expression in fish embryos

The importance of facilitative glucose transporters for fish remains undefined. To study its functional properties, we expressed the putative glucose transporter OnmyGLUT1 in Xenopus laevis oocytes. This heterologous expression system has been very useful for characterizing mammalian and protozoan hexose transporters. The relative abundance of OnmyGLUT1 transcripts in rainbow trout embryos suggested that this protein might play an important role in development. In mammals, GLUT1 is referred to as an early isoform because it is expressed at high levels in embryos and foetuses, being gradually substituted for other GLUT types during the course of development (Santalucia et al., 1992; Postic et al., 1994). We have analysed the expression of OnmyGLUT1 at a number of developmental stages (blastula, early and late gastrula, and during somitogenesis and the formation of the vitelline plexus) using whole-mount in situ hybridization. We also measured
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Synaptic transmission in neurons that express the Drosophila
atypical soluble guanylyl cyclases, Gyc 89Da and Gyc 89Db, is necessary for
the successful completion of larval and adult ecdysis

Synaptic transmission in neurons that express the Drosophila atypical soluble guanylyl cyclases, Gyc 89Da and Gyc 89Db, is necessary for the successful completion of larval and adult ecdysis

Our previous studies using whole-mount in situ hybridization labeling in embryos showed that all three atypical sGC subunit genes, Gyc-89Da, Gyc-89Db and Gyc-88E, were expressed in a subset of peripheral and central neurons (Langlais et al., 2004; Morton et al., 2005a). By examining the number and position of these cells we concluded that these three genes were co-expressed in many of the peripheral sensory neurons (Langlais et al., 2004; Morton et al., 2005a). To better define these co-expression patterns, we used upstream regions of each gene to drive expression of GFP in transgenic flies (designated p89Da-GFP and p89Db-GFP). The portions of genomic DNA 5 ⬘ to Gyc-89Da and Gyc-89Db (1.9 and 3.4·kb, respectively) were sufficient to drive GFP expression in embryos in a pattern that closely resembled that seen with in situ hybridization (Fig.·1). By contrast, however, a 5·kb length of genomic DNA 5 ⬘ to Gyc-88E was insufficient to drive expression of GFP in any cells. Peripheral neurons that expressed GFP in p89Da-GFP and p89Db-GFP larvae included a small subset of cells in the dorsal and terminal ganglia (Fig.·1G–I), which innervate the olfactory and gustatory organs, respectively (Stocker, 1994; Heimbeck et al., 1999), neurons innervating external sensilla along the lateral body wall (Fig.·1D–F) and neurons that innervate the terminal sensory organs (Fig.·1J–L) (Langlais et al., 2004; Morton et al., 2005a). These expression patterns closely matched those seen using in situ hybridization in late embryos (Langlais et al., 2004) (Fig.·1).
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Toxic Eects of Paclobutrazol on Developing Organs at Di erent Exposure Times in Zebrafish

Toxic Eects of Paclobutrazol on Developing Organs at Di erent Exposure Times in Zebrafish

Figure 4. Intestine defects are more severe when exposure to PBZ begins at early stages. Zebrafish embryos were exposed to a range of PBZ concentrations (0.34, 3.4, and 17 µM) beginning at different stages (24, 36, 48, 60, 72, and 96 hpf). Embryos were collected at 5 dpf and analyzed by whole-mount in situ hybridization with an ifapb antisense riboprobe; ifapb is specifically expressed in intestine. The intestinal phenotype was categorized as (A) ‘Normal’, ‘Mild’ defects or ‘Severe’ defects. Images were taken from ventral view. (B) Frequency of intestine phenotypes was compared in zebrafish embryos treated with different concentrations of PBZ at different exposure stages. Data represent the mean of three independent experiments; 30 embryos were assessed in each treatment (N = 3. n = 90 embryos).
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FGFR1 function at the earliest stages of mouse limb development plays an indispensable role in subsequent autopod morphogenesis

FGFR1 function at the earliest stages of mouse limb development plays an indispensable role in subsequent autopod morphogenesis

PD axis (Fig. 7A-D) and an increased length along the DV axis (Fig. 7C) only in the posterior region. These phenotypes started at E10.75 and were obvious by E11.5 with some variations. Whole-mount in situ hybridization using Hoxd12 revealed a normal appearance of the first two or three digits while the development of the posterior digits was abnormal (Fig. 7E, and not shown). These data indicate that deletion of FGFR1 does not affect anterior digit formation despite Cre activity having spread through the entire distal portions of the limb prior to digit formation (Fig. 4G). Of note, the areas showing abnormalities in mutant embryos examined from E9.5-E12.5 (n>30) were always smaller than the ␤ -gal positive areas in reporter mice. This indicates the abnormalities only occur in the region where Hoxb6-Cre is first expressed, i.e. prior to or during the initial budding, and not in the more anterior regions, to where Cre expression spreads by slightly later stages. This is consistent with what was observed in Fgfr1 Co/Co ;AP-2Cre mice.
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Novel bacteriocyte-associated pleomorphic symbiont of the grain pest beetle Rhyzopertha dominica (Coleoptera: Bostrichidae)

Novel bacteriocyte-associated pleomorphic symbiont of the grain pest beetle Rhyzopertha dominica (Coleoptera: Bostrichidae)

specifically in adult males, the bacteriomes and the symbi- otic bacteria therein tend to be atrophied and degenerated [35, 36]. Our whole-mount in situ hybridization data gener- ally agreed with the previous reports, in that most adult males exhibited degeneration of the bacteriomes, although we found that some adult females also showed degeneration of the bacteriomes to some extent (Fig. 7). Notably, similar male-specific symbiont degeneration has been reported from diverse insects, including aphids [53–57], mealybugs [58], stictococcid scales [59], bird lice [60], weevils [61], etc., which may be relevant to vertical symbiont transmission in the host matrilines and/or sex-related differences in the host’s physiological dependency on the symbiont. Consider- ing that only adult females are involved in vertical symbiont transmission to the offspring, it may be unnecessary for adult males to maintain this otherwise costly endosymbiotic system. Since adult females require much more energy and resources for producing eggs than adult males do for producing sperm, symbiont-provisioned nutrients such as amino acids and vitamins might be essential for adult females but not for adult males.
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Physiological effects of KDM5C on neural crest migration and eye formation during vertebrate development

Physiological effects of KDM5C on neural crest migration and eye formation during vertebrate development

Molecular analyses of KDM5C functional roles using whole‑mount in situ hybridization, β ‑ galactosidase staining, and reverse transcription‑polymerase chain reaction revealed that los[r]

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Third generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

Third generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

subcellular resolution within whole-mount vertebrate embryos (Trivedi et al., 2018). Precision increases with probe set size (Trivedi et al., 2018), so the prospect of using large unoptimized split-initiator probe sets is highly appealing. To test mRNA relative quantitation with automatic background suppression, we redundantly detected target mRNAs using two split-initiator probe sets each triggering a different spectrally distinct HCR amplifier (Fig. 5AB). If HCR signal scales approximately linearly with the number of target mRNAs per voxel, a two-channel scatter plot of normalized voxel intensities will yield a tight linear distribution with approximately zero intercept. Conversely, observing a tight linear distribution with approximately zero intercept (Fig. 5C), we conclude that the HCR signal scales approximately linearly with the number of target mRNAs per imaging voxel, after first ruling out potential systematic crowding effects that could permit pairwise voxel intensities to slide undetected along the line (Figs S13 and S23). Using 20 unoptimized split-initiator probe pairs (v3.0) per channel, the observed accuracy (linearity with zero intercept) and precision (scatter around the line) are both excellent for subcellular 2.1×2.1×2.7 µm voxels within a whole-mount chicken embryo. Just as quantitative PCR (qPCR) enables analog mRNA relative quantitation in vitro (Gibson et al., 1996; Heid et al., 1996), qHCR imaging enables analog mRNA relative quantitation in situ.
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Evolutionary loss of melanogenesis in the tunicate Molgula occulta

Evolutionary loss of melanogenesis in the tunicate Molgula occulta

Pigment cell marker expression in Molgula embryos We next sought to determine the spatiotemporal expres- sion pattern of selected M. occulta and M. oculata Tyrosinase family genes, by whole-mount mRNA in situ hybridization on embryos at different developmen- tal stages (Fig.  2). In the larvae of M. occulta, bilateral Mooccu.Tyrp.a expression was detected in embryos start- ing at 10  h post-fertilization (hpf), which is around the time they are hatching (Fig. 2a). Their number and loca- tion in the embryo suggest they correspond to vestigial pigment cell precursors (a9.49 lineage in Ciona robusta, Fig. 2b). In contrast, Mooccu.Tyr is absent from the tran- scriptome (see above) [39]. Indeed, mRNA in situ hybrid- ization using a riboprobe prepared from a genomic clone of the Mooccu.Tyr locus (see below) showed no tran- scriptional activity of this sequence during development nor in hatched larvae (Fig.  2d, e). This further confirms the complete loss of transcriptional activity of this gene in M. occulta as suggested by the RNAseq data. Taken together, these data suggest that, although unpigmented M. occulta specify vestigial pigment cell precursors that transcribe Mooccu.Tyrp.a, the rate-limiting melanogenic gene Mooccu.Tyr is not expressed in these cells.
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FGF signaling is required for initiation of feather placode development

FGF signaling is required for initiation of feather placode development

soaked in FGF10 (referred to as FGF10 beads) to ppf explants and assayed for changes in gene expression normally associated with the onset of bud development. In explants cultured in the presence of FGF10 beads, expression of Bmp2 was induced close to the bead (Fig. 7B). Likewise, FGF10 beads also induced an up-regulation of Wnt6 expression (Fig. 7D), as normally observed in the ectoderm of nascent feather buds. A continuous stripe of follistatin expression normally precedes the localized expression of the earliest bud markers, labeling the regions where the next feather placodes will form. Subsequently, follistatin expression is transiently detected in the placodes, but is then maintained in a ring along the edge of each placode (Ohyama et al., 2001; Patel et al., 1999). In ppf explants cultured with FGF10 beads, upregulated expression of follistatin was detectable around the beads (Fig. 7E). PBS beads in contrast had no effects on the expression of any of the three markers (Fig. 7A,C,D). Thus, FGF10 is sufficient to induce changes in ectodermal gene expression characteristic of the initiation of bud development. Notably, while upregulating marker gene expression, FGF10 beads also blocked the formation of individual feather buds in their vicinity, similar to what has been described by Tao et al. (Tao et al., 2002). During development of the feather array, bud spacing is thought to be controlled through positive feedback Fig. 5. Expression of Fgf3 and Fgf10 in early feather development. Detection of Fgf3 (A- C) or Fgf10 (D-F,J-L) expression by whole- mount RNA in situ hybridization of embryos at HH28, before the onset of feather placode development, and at HH30. (C,F) Expression of Fgf3 and Fgf10 in embryos infected with RCAS-FGFR1-Fc and RCAS-FGFR2-Fc at HH18. The nude region devoid of Fgf3 and Fgf10 expression is included with a bracket. (G-I) Vibratome sections, at the indicated positions, through the embryos shown in B, D and E, respectively. Arrowheads in D indicate the stripes of Fgf10 expression in the region where bud development will be initiated. (J-L) Expression of Fgf10 in the alar (J), pectoral (K) and tail tract (L). Arrows indicate the feather tract, delineated by Fgf10 expression. nt, neural tube.
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Mapping a multiplexed zoo of mRNA expression

Mapping a multiplexed zoo of mRNA expression

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin- embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.
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Molecular cytogenetic identification of three rust-resistant wheat-Thinopyrum ponticum partial amphiploids

Molecular cytogenetic identification of three rust-resistant wheat-Thinopyrum ponticum partial amphiploids

Background: Thinopyrum ponticum (2n = 10× = 70, J S J S J S J S JJJJJJ) is an important wild perennial Triticeae species that has a unique gene pool with many desirable traits for common wheat. The partial amphiploids derived from wheat-Th. ponticum set up a bridge for transferring valuable genes from Th. ponticum into common wheat. Results: In this study, genomic in situ hybridization (GISH), multicolor GISH (mcGISH) and fluorescence in situ hybridization (FISH) were used to analyze the genomic constitution of SN0389, SN0398 and SN0406, three octoploid accessions with good resistance to rust. The results demonstrated that the three octoploids possessed 42 wheat chromosomes, while SN0389 contained 12 Th. ponticum chromosomes and SN0398 and SN0406 contained 14 Th. ponticum chromosomes. The genomic constitution of SN0389 was 42 W + 12J S , and for SN0398 and SN0406 it was 42 W + 12J S + 2 J. Chromosomal variation was found in chromosomes 1A, 3A, 6A, 2B, 5B, 6B, 7B, 1D and 5D of SN0389, SN0398 and SN0406 based on the FISH and McGISH pattern. A resistance evaluation showed that SN0389, SN0398 and SN0406 possessed good resistance to stripe and leaf rust at the seedling stage and adult-plant stage.
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The reliability of rabbit monoclonal antibodies in the immunohistochemical assessment of estrogen receptors, progesterone receptors, and HER2 in human breast carcinomas

The reliability of rabbit monoclonal antibodies in the immunohistochemical assessment of estrogen receptors, progesterone receptors, and HER2 in human breast carcinomas

clinical analysis of HER2 gene amplification status. In the current study, FISH and a chromogen-based in situ hybridization system (SISH) were compared with each other and with each of the antibody assays investigated. It is interesting that the highest concordance rates between the immunohistochemical assays and in situ–based systems were between the rabbit monoclonal antibodies and FISH. Arguably, the most reliable results were obtained with FISH and the rabbit monoclonal antibody 4B5, as all 3+ cases with this antibody gave HER2 gene amplification by FISH (plus 1 equivocal result), while all 0 and 1+ cases showed no HER2 gene amplification.
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