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Acid phosphatase detection using fast blue or fast black reagent is routinely performed at the RCMP National Forensic Laboratory Services as a presumptive test for semen. In training exercises, sam...

Neocortical layer (L) 1 is a locus for interactions between long-range inputs, L1 interneurons and apical tuft dendrites of pyramidal neurons. Even though we have a wealth of information about L1, the level and effect of local input to this layer have not been quantified. Here we characterized the input to L1 of mouse somatosensory cortex with fast blue, monosynaptic rabies and optogenetics. Our work shows that most of the input to L1 is local, and that both local and long-range inputs to this layer arise predominantly from L2/3 and L5 neurons. Subtypes of L5 and L6b neurons project to the overlying L1 with different probabilities. VIP and SST interneurons in L2/3 and L5 also innervate L1. A subset of local L5, the intratelencephalic, pyramidal neurons, drive L1 interneurons but have no effect on L5 apical tuft dendrites. Monosynaptic rabies-based retrograde labelling reveals presynaptic boutons covering the entire somato-dendritic axis of pyramidal neurons, including in L1. When fast blue application was combined with rabies virus, we found that only a fraction of local and long-range neurons was both presynaptic to L5 neurons and projected to L1. These results demonstrate that L1 receives a large proportion of its input from local neurons, and that some of these inputs specifically target interneurons. We conclude that L1 is not just a site for interaction between long-range feedback and apical tuft dendrites of pyramidal cells, it is also a site for complex modulation of pyramidal neurons and interneurons by local inputs.

AT2018cow is a unique transient that stands out due to its relatively fast light-curve, high peak bolometric luminosity, and blue color. These properties distinguish it from typical radioactively powered core-collapse supernovae (SNe). Instead, the characteristics are more similar to a growing sample of Fast Blue Optical Transients (FBOTs). Mostly discovered at hundreds of Mpc, FBOT follow-up is usually limited to several photometry points and low signal-to-noise spectra. At only ~60 Mpc, AT2018cow offers an opportunity for detailed followup. Studies of this object published to date invoke a number of interpretations for AT2018cow, but none of these specifically consider the interacting Type Ibn SN subclass. We point out that while narrow lines do not dominate the spectrum of AT2018cow, as narrow Balmer lines typically do in SNe IIn, the narrow lines in AT2018cow may nevertheless be a mix of unresolved HII region emission and emission from slow, pre-shock CSM. We compare AT2018cow to interacting SNe Ibn and IIn and find a number of noteworthy similarities, including light-curve rise and fall times, peak magnitude, X-ray light-curves, and spectroscopic properties. In particular, the He I lines in AT2018cow closely resemble those in some examples of SNe Ibn or transitional SNe Ibn/IIn objects. We therefore explore the hypothesis that CSM interaction in a relatively H-poor system might have some merit in explaining observed properties of AT2018cow, and we go on to consider progenitor implications for AT2018cow, FBOTs, and SNe~Ibn., Comment: 11 pages, 9 figures, submitted to MNRAS

A paired electrochemical synthesis of new phenylcarbonimidoyl dicyanide dyes was performed by the electrooxidation of fast violet B (FVB) and fast blue BB (FBB) in the presence of malononitrile. Ou...

The phenolic compounds of extra-virgin olive oil (EVOO) are key contributors of nutritional and sensory quality as well as chemical stability. The reference method for their determination is the HPLC-UV, which is cost-/time-expensive. In this work, total phenolic compounds were evaluated in unfractionated EVOO adapting the Fast Blue BB (FBBB) assay, which involves the spectrophotometric (absorbance at 420 nm) determination of azo derivatives resulting from the coupling of phenolic compounds with FBBB diazonium salt in alkali pH. When tested on 26 EVOO samples, the FBBB assay and HPLC-determinations were strikingly correlated (R2 = 0.9653), differently from FBBB and Folin-Ciocalteu assays, which showed poor correlation. The assay is simple, repeatable, robust, rapid and cheap, and results might be evaluated on a printed colorimetric scale. This protocol of the FBBB assay could be routinely used to categorize EVOO according to the health claim allowed by EFSA concerning the content of phenolic compounds.

Celiac disease is an immune-mediated systemic disorder elicited by gluten and related prolamines in genetically susceptible individuals. The current treatment is a strict and lifelong gluten-free diet. However, compliance with the gluten-free diet is not always adequate and many food products contain low concentrations of gluten. The determination of dietary transgressions is a challenge for patients, physicians and dietitians. Alkylresorcinols (AR) have been proposed as sensitive and specific biomarkers of gluten consumption. In this work silica-polymer composites doped with fast blue B reagent (FB) have been used to estimate alkylresorcinols in biological samples. The proposed colorimetric device was synthetized by immobilizing FB into polydimethylsiloxane-tetraethylortosilicate composite. The assay was based on the spontaneous release of FB to the solution con-taining AR (3,5-dihydroxyhydrocinnamic acid, DHCA) and the azocomplexe formation (520 nm). The response was evaluated with UV-vis spectroscopy and chromatography (in-tube SPME-Capillary LC-DAD) to isolate DHCA signal. The spectroscopic analysis can be used as a screening tool to differentiate positive and negative samples. Meanwhile, the chromatographic assay was necessary to isolate DHCA response in positive samples. LOD was 60 ng mL-1 by adding a SPE step. Precision provided adequate results (RSD < 15%). Preliminary studies in urine samples displayed successful results, showing DHCA can be used as biomarker of gluten intake. The here pro-posed methodology clearly simplifies the dietary transgression evaluation thanks to the development of a pre -screening tool before the chromatographic analysis.

Folin-Ciocalteu and the more recent Fast Blue BB (FBBB) reactions are used for the quantification of total phenolics in food matrices. Despite its known interferences, Folin-Ciocalteu is still widely employed, considering Solid Phase Extraction (SPE) as clean-up step only in a few cases. Meanwhile, FBBB has shown no interferences for the determination of total phenolics in fruits and cereals, although its utilization in other popular matrices containing potential interferences, such as legumes, remains unexplored. In this study, the total phenolic content of 24 flours from legumes, cereals, fruits, nuts and plant seeds was evaluated by Folin-Ciocalteu and FBBB, with and without SPE. Folin-Ciocalteu showed interferences for 75% of the flours (attributed to reducing sugars and enediols), whereas FBBB only for legumes and nuts (attributed to the presence of tyrosine), both methods in those matrices requiring SPE. Although both SPE-FBBB and SPE-Folin-Ciocalteu presented excellent reproducibility, SPE-FBBB displayed 1.5 times higher sensitivity.

DES Collaboration: et al., Rapidly evolving transients (RETs), also termed fast blue optical transients, are a recently discovered group of astrophysical events that display rapid luminosity evolution. RETs typically rise to peak in less than 10 d and fade within 30, a time-scale unlikely to be compatible with the decay of Nickel-56 that drives conventional supernovae (SNe). Their peak luminosity spans a range of −15 < Mg < −22.5, with some events observed at redshifts greater than 1. Their evolution on fast time-scales has hindered high-quality follow-up observations, and thus their origin and explosion/emission mechanism remains unexplained. In this paper, we present the largest sample of RETs to date, comprising 106 objects discovered by the Dark Energy Survey, and perform the most comprehensive analysis of RET host galaxies. Using deep-stacked photometry and emission lines from OzDES spectroscopy, we derive stellar masses and star formation rates (SFRs) for 49 host galaxies, and metallicities ([O/H]) for 37. We find that RETs explode exclusively in star-forming galaxies and are thus likely associated with massive stars. Comparing RET hosts to samples of host galaxies of other explosive transients as well as field galaxies, we find that RETs prefer galaxies with high specific SFRs (〈log (sSFR)〉 ∼ −9.6), indicating a link to young stellar populations, similar to stripped-envelope SNe. RET hosts appear to show a lack of chemical enrichment, their metallicities akin to long-duration gamma-ray bursts and superluminous SN host galaxies (〈12 + log (O/H)〉 ∼ 9.4). There are no clear relationships between mass or SFR of the host galaxies and the peak magnitudes or decline rates of the transients themselves., We acknowledge support from STFC grant ST/R000506/1. MSm, MSu, and CPG acknowledge support from from the European Union’s 7th Framework Programme (EU/FP7) European Research Council (ERC) grant no. 615929. LG was funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 839090. This work has been partially supported by the Spanish grant PGC2018-095317-B-C21 within the European Funds for Regional Development (FEDER). LK was supported by the Science and Technology Facilities Council (grant number ST/P006760/1) through the DISCnet Centre for Doctoral Training. The DES data management system is supported by the National Science Foundation under Grant Numbers AST-1138766 and AST-1536171. The DES participants from Spanish institutions are partially supported by MINECO under grants AYA2015-71825, ESP2015-66861, FPA2015-68048, SEV-2016-0588, SEV2016-0597, and MDM-2015-0509, some of which include ERDF funds from the European Union. IFAE is partially funded by the CERCA program of the Generalitat de Catalunya. Research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013) including ERC grant agreements 240672, 291329, and 306478. We acknowledge support from the Brazilian Instituto Nacional de Ciencia e Tecnologia (INCT) e-Universe (CNPq grant 465376/2014-2)

espanolEl objetivo de este estudio fue cuantificar los compuestos fenolicos totales de diferentes granos ancestrales: dos variedades de tef (Eragrostis tef), trigo sarraceno (Fagopyrum esculentum) y trigo espelta (Triticum spelta) por diferentes metodos espectrofotometricos (Folin-Ciocalteu y Fast Blue BB) y comparar con los resultados obtenidos de estudiar la composicion fenolica individualizada mediante cromatografia liquida de alta resolucion acoplada a un sistema de deteccion de fotodiodos y espectrometria de masas (HPLC-DAD-MS). Tambien se determino la actividad antioxidante in vitro de los extractos de los granos ancestrales estudiados por el metodo TEAC (capacidad antioxidante equivalente de Trolox) y FRAP (poder antioxidante reductor del hierro). El uso de HPLC-DAD-MS permitio identificar un gran numero de compuestos fenolicos: compuestos hidroxibenzoicos, hidroxicinamicos, flavonoles, flavonas y flavanoles. Las flavonas fueron la familia de compuestos fenolicos mas abundante detectada en las muestras de espelta, teff, blanco y marron. Sin embargo, en el trigo sarraceno son los flavanoles (64 %). La cuantificacion de los compuestos fenolicos por HPLC-DAD-MS parece mas oportuna que por los metodos espectrofotometricos. Con respecto a la actividad antioxidante, el trigo sarraceno mostro los valores mas altos frente a su capacidad de captar radicales libres y poder reductor en relacion a las otras muestras estudiadas, pudiendose relacionar con su alto contenido en compuestos fenolicos. EnglishThe aim of this study was to quantify the total phenolic compounds of different ancestral grains such as two varieties of tef (Eragrostis tef) white and brown, buchwheat (Fagopyrum esculentum) and spelta (Triticum spelta), using different spectrophotometric methods (Folin-Ciocalteu and Fast Blue BB) and to compare with the obtained results using high performance liquid chromatography coupled to diode array detector and mass spectrometry (HPLC-DAD-MS). Furthermore, antioxidant activity of the extracts from these samples has been compared using two different in vitro assays: ferric reducing power (FRAP) and the ability to scavenge the ABTS•+. The use of HPLC-DAD-MS allowed us to detect different groups of phenolic compounds: hydroxybenzoic and hydroxycinnamic derivatives, flavonols, flavones and flavanols. Flavones derivatives were the most abundant in spelta, white and brown teff. However, buckwheat presented the highest in flavanols (64 %). The phenolic compounds quantification carried out by HPLC-DAD-MS could be more appropriate than the obtained results using spectrophotometric methods. Regarding the antioxidant activity, the buckwheat seeds showed the highest value, which could be related to the highest phenolic content.

The Fast Blue BB (FBBB) colorimetric test has been shown to differentiate between the different cannabinoids found in cannabis addressing the urgent need to distinguish between hemp-type cannabis, which is legal in every state of the US, from marijuana-type cannabis (≥0.3% w/w THC). FBBB forms a red chromophore in the presence of THC, an orange chromophore in the presence of CBD, a detectable fluorophore with THC but no fluorescence with CBD. We report, for the first time, a miniaturized reaction directly on a 3.5 mm diameter solid substrate of a material previously developed in our laboratory using a methanol extract from ∼10 mg of plant sample. Different cannabinoids, various herbs and spices, and authentic cannabis samples were tested with the optimized color test, and the FBBB reaction was found selective for THC relative to other cannabinoids and herbs and spices. RGB (Red, Green, Blue) numerical codes were obtained for each color (and fluorescence) image produced by the reaction. Linear Discriminant Analysis (LDA) results correctly classify a set of 25 known plant samples (marijuana, hemp and herbs and spices) as either containing THC levels below 0.3% (w/w) (hemp) or as containing high THC levels (≥0.3% w/w) and low CBD levels (marijuana). Cannabis samples containing low THC (but just above 0.3%) and high CBD (THC:CBD ratios

Strawberry cultivars Albion, Aromas, Camarosa, Camino Real, Monte Rey, Portola, and San Andreas were evaluated for their antioxidant potential. Anthocyanins cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, and pelargonidin-O-rutinoside were identified by UPLC-PDA-ESI/MS2 in all cultivars studied. Total anthocyanin content (TAC) measured by HPLC/DAD ranged from 15.67 mg/100 g in Camino Real to 27.62 mg/100 g in Camarosa. TAC measured by the pH-differential method was strongly correlated with results from HPLC analysis (r = 0.82). Cultivar Monte Rey showed the highest total phenolic content (TPC), with 2.48 mg/g measured by the Folin-Ciocalteu (FC) method. A strong correlation was observed between TPC measured by the FC and the Fast Blue BB methods (r = 0.71). DPPH radical scavenging capacity (EC50) showed a weak correlation with TPC (r = −0.48) but a strong correlation with TAC (r = −0.93), which indicates that the antioxidant potential of strawberry cultivars can be attributed mainly to their anthocyanin content.

Sympathetic innervation of the stomach was studied in rats by the method of retrograde axon transport of Fast Blue in postnatal ontogenesis. The number of labeled neurons increased in the first 10 days of life and then did not change until the senescence. All labeled neurons innervating the stomach contain the catecholamine synthesis enzyme, tyrosine hydroxylase. The proportion of labeled neuropeptide Y-immunopositive neurons did not change in the development, the percentage of labeled calbindin-immunoreactive neurons decreased in the first month of life.

Neuro-tracing approach is a great option to study innervation of the visceral organs including the kidneys. Important factors contributing to the success of this technique include the choice of a neuro-tracer, and delivery methods to result in successful labeling of peripheral sensory and motor ganglia. The neuro-tracer is usually applied directly to the kidney accessed via a surgical opening of the abdominal wall under deep anesthesia. A series of local microinjections of the dye are performed followed by a wound closure, and recovery period from the surgery. An extra care should be taken to prevent neuro-tracer spillage and accidental labeling of the surrounding organs during injections of the dye. Retrograde neuro-tracers like Fast Blue do not cross synapses, therefore, only neuronal bodies located within dorsal root ganglion neurons and major peripheral ganglia will be labeled by this approach. Retrogradely labeled peripheral neurons could be freshly isolated and dissociated for electrophysiological recordings and biochemical analyses (gene and protein expression), whereas the whole fixed ganglia could be sectioned to undergo immunohisto- and immunocytochemical targeted staining.

The autonomic nervous system is a sophisticated and independent structure composed of two antagonistic (opposing) divisions (sympathetic and parasympathetic) that control many vital functions including: homeostasis maintenance, heart rate, blood circulation, secretion, etc. Thymus is one of the most important primary lymphoid organs playing a role in the developing of a juvenile’s immune system mainly by maturation, development, and migration of T-cells (T lymphocytes). In the last decades, several studies identifying sources of the thymic autonomic supply have been undertaken in humans and several laboratory rodents but not in higher mammals such as the pig. Therefore, in the present work, retrograde tracing technique of Fast Blue and DiI was used to investigate the sources of sympathetic efferent supply to the porcine thymus. After Fast Blue injection into the right lobe of the thymus, the presence of Fast Blue-positive neurons was found in the unilateral cranial cervical ganglion (82.8 ± 3.0% of total Fast Blue-positive neurons) as well as in the middle cervical ganglion (17.2 ± 3.0%). Injection of DiI resulted in the presence of retrograde tracer in neurons of the cranial cervical ganglion (80.4 ± 2.3% of total amount of DiI-labelled neurons), the middle cervical ganglion (18.4 ± 1.9%), and the cervicothoracic ganglion (1.2 ± 0.8%). The present report provides the first data describing in details the localization of primary efferent sympathetic neurons innervating the porcine thymus.

The last ten years have opened up a new parameter space in time-domain astronomy with the discovery of transients defying our understanding of how stars explode. These extremes of the transient paradigm represent the brightest - called superluminous supernova - and the fastest - known as fast, blue optical transients - of the transient zoo. The number of their discoveries and information gained per event have witnessed an exponential growth that has benefited observational and theoretical studies. The collected dataset and the understanding of such events have surpassed any initial expectation and opened up a future exploding with potential, spanning from novel tools of high-redshift cosmological investigation to new insights into the final stages of massive stars. Here, the observational properties of extreme supernovae are reviewed and put in the context of their physics, possible progenitor scenarios and explosion mechanisms., Comment: Author's version of invited Review published as part of a Supernova Focus Issue in Nature Astronomy; 26 pages, 4 Figures, 1 Table

Cannabis is one of the most abused illicit drugs worldwide while also finding more acceptance for medical and recreational use. Presumptive testing of suspected drug seizures using colorimetric spot tests is an important fast and efficient preliminary step in the identification of drug classes such as the cannabinoids. This study describes the chemical reactions between the Fast Blue BB (FBBB) reagent and three cannabinoids (Δ9-THC, CBD, and CBN) when using a small polymer strip (50 ng LOD) and when using a new capillary microextraction of volatiles (CMV) sorbent device (500 ng LOD). The product chromophore when reacting FBBB and THC was isolated and identified, for the first time, by HNMR and confirmed by accurate mass spectrometry. The coupling of FBBB to the para position (versus the ortho position) relative to the phenolic group of Δ9-THC was expected, and 1H NMR studies and an ab initio reaction coordinate investigation at the B3LYP/3-21g level of theory provide computational support for these findings.

The new, selective and sensitive visible spectrophotometric method has been developed for the estimation of Esomeprazole in bulk and in pharmaceutical preparations. This method is based on the reaction with wool fast blue, in the presence of buffer at PH 1.5 to form a colored species with a I»max 590nm. Beer’s law is obeyed in the concentration range of 50-250µg/ml for both the methods. The method was extended to pharmaceutical formulations and there was no interference form in any common pharmaceutical excipients and diluents. The result of analysis has been validated statistically and by recovery studies. Keywords: Spectrophotometric determination Wool fast blue, Esomeprazole.

In primates, the koniocellular (K) layers of the dorsal lateral geniculate nucleus (LGN) and the calbindin-rich subdivisions of the inferior pulvinar (IPul) nucleus are considered part of a thalamic matrix system which projects diffusely to superficial cortical layers. Activity in the matrix system is proposed to coordinate oscillatory activity in thalamocortical loops. Further, since both K cells and IPul are involved in visual processing pathways, especially in alternative pathways to visual cortex after V1 lesion in early life (“blindsight”), their functional similarities have been strongly implicated. Here we tested the hypothesis that calbindin-positive K cells and IPul cells constitute a continuous group of cells. By combining immunohistochemistry and a high-throughput neuronal tracing method, we found that both K cells and IPul form reciprocal connections with striate and extrastriate cortices; whereas principal laminae of LGN do not receive inputs from extrastriate cortex and only project sparsely to these areas. Retrograde labelled cells in lateral division of IPul merged seamlessly into the retrograde labelled cells in K layers. These results supported the continuity between LGN K layers and IPul, providing the anatomical basis for functional congruity of this part of dorsal thalamic matrix.

There is a strong need for simple and fast methods for wound infection determination. Myeloperoxidase, an immune system-derived enzyme was found to be a suitable biomarker for wound infection. Hence, alkoxysilane-derivatized Fast Blue RR was immobilized via simple hydrolytic polymerization. The resulting enzyme-responsive siloxane layers were incubated with myeloperoxidase, wound fluid or hemoglobin. The reaction was monitored via HPLC measurements and the color development quantified spectrophotometrically. Myeloperoxidase was indeed able to oxidize immobilized Fast Blue RR leading to a blue colored product. No conversion was detected in non-infected wound fluids. The visible color changes of these novel materials towards blue enable an easy distinction between infected and non-infected wound fluids.

A histological method for detecting of 5-n-alkylresorcinols in grains sections using the staining with Fast Blue B was developed. Taking advantage of that method it was found that alkylresorcinols are localized exclusively in the outer cuticle, but both germs and starchy endosperm of rye and wheat seeds are entirely free of these compounds.

The Cannabis sativa L. plant is a species rich in a variety of cannabinoid compounds and Δ9-tetrahydrocannabinol (Δ9-THC) has been reported as a main psychotropic substance. In this study, electrospray ionization (ESI), coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), was used in order to perform a direct and fast analysis of street marijuana samples. ESI(−)-FT-ICR MS detected 21 cannabinoid species in the deprotonated form, [M − H]−. Other species were detected in regions of m/z 600–800 and 800–1000, corresponding to dimers and trimers of cannabinoids. In addition, ESI(−) was better able to analyze the chemical profile of terpenophenolic species (CcHhOo) than ESI(+). ESI(+)-FT-ICR MS detected the presence of adulterants such as cocaine, lidocaine, and nicotine. Finally, the sensitivity of fast blue B colorimetric testing was also evaluated and the results were compared to the ESI(−)FT-ICR MS data.

Polyphenols are bioactive substances of vegetal origin with a significant impact on human health. The assessment of polyphenol intake and excretion is therefore important. The Folin–Ciocalteu (F–C) method is the reference assay to measure polyphenols in foods as well as their excretion in urine. However, many substances can influence the method, making it necessary to conduct a prior cleanup using solid-phase extraction (SPE) cartridges. In this paper, we demonstrate the use of the Fast Blue BB reagent (FBBB) as a new tool to measure the excretion of polyphenols in urine. Contrary to F–C, FBBB showed no interference in urine, negating the time-consuming and costly SPE cleanup. In addition, it showed excellent linearity (r2 = 0.9997), with a recovery of 96.4% and a precision of 1.86–2.11%. The FBBB method was validated to measure the excretion of polyphenols in spot urine samples from Spanish children, showing a good correlation between polyphenol intake and excretion.

Dudek, A., Sienkiewicz, W. and Kaleczyc, J. 2014. Contribution of the dorsal branch of the accessory nerve to the innervation of the trapezius muscle in the pig – ar etrograde tracing study —Acta Zoologica (Stockholm) 00: 000–000. The study was aimed to establish the contribution of the dorsal branch of the accessory nerve (DBXI) to the innervation of the porcine trapezius muscle (TRAP). Combined retrograde tracing using fluorescent tracer Fast blue (FB) and surgical denervation procedure (excision of DBXI segment) were applied. FB+ neurons supplying the cervical (c-TRAP) and thoracic part (th-TRAP) of TRAP were localized in following nerve centres: the ipsilateral ventral horn of the grey matter of cervical neuromers, ipsilateral spinal ganglia and bilateral sympathetic chain ganglia. After the excision of DBXI segment, no FB+ motoneurons supplying c-TRAP were found while the mean number of those supplying th-TRAP was significantly decreased. A slight decrease in average numbers of sensory and autonomic neurons implemented in the innervation of both parts of TRAP was also observed. This study has revealed that in the pig DBXI is the only motor pathway to c-TRAP while the vast majority of motoneurons supplying porcine th-TRAP send their axons via DBXI.

Response surface methodology was used to optimize the total phenolic content (TPC) result in white rice, using a new methodology that employed Fast Blue BB (FBBB) reagent, which reacts more specifically with phenolic compounds, along with direct QUENCHER procedure. This is the first time in which such conciliation between FBBB reagent and QUENCHER was done. The obtained model allowed to evaluate how the factors influenced the method linearity (R2), improving the choice of the optimal system condition. Furthermore, the similarity between experimental results and values generated by the model indicated the method validity within the studied region. Thus, the optimal condition determined in the present work was 30 min reaction time, 0.17 mL FBBB reagent and use of 5% NaOH as the catalyst for the reaction. This optimized condition suggested that the combination of the method, which used FBBB reagent and the direct QUENCHER procedure, was rapid and effective, yielding high TPC values (1488.73 mg of gallic acid equivalent per 100 g).

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.