Wir danken Jayne Selegue, Margaret Hollingsworth, Jin Berry, Ryo Futahashi, Najmus Sahar Mahfooz, Aleksandar Popadic, Bob Reed, Roche Technical Support, um Hilfe bei der Fehlersuche dieses Protokoll. Wir danken auch William Piel um Rat bei der Bearbeitung des Films.

TAG 1 <ol><li> Bereiten Sie die folgenden Lösungen: <ul><li> 10x PBS </li><li> 1x PBS </li><li> 1x PBT </li><li> ddH 2 O <ul><li> Add DEPC für 0,1% Gesamtvolumen und schütteln kräftig Lösungen. </li><li> Autoclave. </li></ul></li><li> 4% Paraformaldehyd Fix - mit PBS-DEPC </li><li> Proteinase K-Lösung - wenn Niederschlag vorhanden ist, Vortex kräftig, bevor Sie aliquoten </li><li> Verdauung Anschlagpuffer </li><li> Prähybridisierungspuffer </li><li> 50:50 PBT: Prähybridisierungspuffer </li><li> Hybridisierungspuffer <ul><li> Lösungen sollten gehalten RNase-frei sein. Verwenden Sie entweder Glas-ware, die gebacken hat (4 Stunden bei 250 ° C) oder Einweg-Kunststoff. Serologische Pipetten sind sehr nützlich für die Herstellung von Lösungen </li></ul></li></ul></li><li> Dissect Flügel von Zeit inszenierte Puppen in PBS </li><li> Bewegen Flügel direkt auf Buffer in Vertiefungen von 24-Well-Kulturplatte Fix bei Raumtemperatur </li><li> Fix-Discs für 2 Stunden </li><li> Wash 5 x 5 Minuten in PBT </li><li> Inkubieren Flügel für 3 Minuten in Proteinase K-Lösung </li><li> Spülen Sie 2 x in der Verdauung Anschlagpuffer </li><li> Wash 5 x 5 Minuten in PBT </li><li> Wash 2 x 5 Minuten in 50:50 PBT: PHB </li><li> Wash 1 x 10 Minuten in PHB </li><li> Inkubieren in PHB mindestens 1 Stunde bei 55 ° C. </li><li> Heat-denaturieren RNA-Sonde (20-50ng pro Well benötigt) (80 ° C für 5 Minuten) und fügen Sie Hybridisierungspuffer (100 ul pro Well benötigt) </li><li> Add-Sonde in die Vertiefungen und inkubieren 48 Stunden bei 55 ° C </li></ol> TAG 3 <ol><li> Wash 4 x 5 Minuten in 55 ° C PHB </li><li> Inkubieren über Nacht in PHB bei 55 ° C </li></ol> TAG 4 <ol><li> Bereiten Sie die folgenden Lösungen: <ul><li> Antikörper Blockpuffer </li></ul></li><li> Wash 1 x 5 Minuten in 50:50 PBT: PHB </li><li> Wash 4 x 5 Minuten in PBT </li><li> Inkubieren Flügeln für 1 Stunde in Blockpuffer bei 4 ° C </li><li> Inkubieren Flügel in einer 1:2000 Verdünnung von Anti-Dig-Antikörper über Nacht bei 4 ° C </li></ol> TAG 5 <ol><li> Bereiten Sie folgende Lösungen: <ul><li> Erkennung Buffer </li></ul><ul><li> Entwickeln Solution - lichtempfindlich (wrap in Folie) </li></ul></li><li> Wash 3 x 5 Minuten in PBT bei Raumtemperatur </li><li> Wash 7 x 5 Minuten in PBT </li><li> Spülen Flügel zwei Mal in Detektionspuffer </li><li> Develop Flügel in 1 ml der Entwicklung Solution - Zeit der Entwicklung muss jedes Mal kontrolliert werden - die Entwicklung von Zeiten können auch geändert werden, wenn die gleichen Sonden-und Entwicklungsländern Lösung - in der Regel nach 5-10 Minuten zu überprüfen und dann zu erhöhen Abständen bis zu übernachten. Platte sollte in Folie gewickelt werden, um Belichtung zu verhindern, wenn nicht die Überprüfung von Proben. </li><li> Spülen Sie fünf Mal in Entwicklungsländern Anschlagpuffer </li><li> Berg Flügel. </li></ol>

<ol>
	<li>Carroll, S. B., Gates, J., Keys, D. N., Paddock, S. W., Panganiban, G. E. F., Selegue, J. E., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+and+eyespot+determination+in+butterfly+wings.">Pattern formation and eyespot determination in butterfly wings.</a> Science. 265, 109-114 (1994).</li><li>Chomczynski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+in+formamide+protects+RNA+from+degradation.">Solubilization in formamide protects RNA from degradation.</a> Nucleic Acids Research. 20, 3791-3792 (1992).</li><li>Keys, D. N., Lewis, D. L., Selegue, J. E., Pearson, B. J., Goodrich, L. V., Johnson, R. J., Gates, J., Scott, M. P., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recruitment+of+a+hedgehog+regulatory+circuit+in+butterfly+eyespot+evolution.">Recruitment of a hedgehog regulatory circuit in butterfly eyespot evolution.</a> Science. 283, 532-534 (1999).</li><li>Marcus, J. M., Ramos, D. M., Monteiro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+line+transformation+of+the+butterfly+Bicyclus+anynana.">Germ line transformation of the butterfly Bicyclus anynana.</a> Proc R Soc Lond B (Suppl). 271, S263-S265 (2004).</li><li>Monteiro, A., Glaser, G., Stockslagger, S., Glansdorp, N., Ramos, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+insights+into+questions+of+lepidopteran+wing+pattern+homology.">Comparative insights into questions of lepidopteran wing pattern homology.</a> BMC Developmental Biology. 6, 52 (2006).</li><li>Ramos, D. M., Kamal, F., Wimmer, E. A., Cartwright, A. N., Monteiro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+control+of+transgene+expresson+using+laser+induction+of+the+hsp70+promoter.">Temporal and spatial control of transgene expresson using laser induction of the hsp70 promoter.</a> BMC Developmental Biology. 6, 55 (2006).</li><li>Weatherbee, S. D., Nijhout, H. F., Grunert, L. W., Halder, G., Galant, R., Selegue, J., Carroll, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrabithorax+function+in+butterfly+wings+and+the+evolution+of+insect+wing+patterns.">Ultrabithorax function in butterfly wings and the evolution of insect wing patterns.</a> 9, 109-115 (1999).</li></ol>

Optimierung bestehender Protokolle In situ Hybridisierungen auf Larven Flügel Scheiben erfolgreich durchgeführt wurden mit Scheiben aus Precis coenia Schmetterlinge (Carroll et al 1994;. Keys et al 1999;. Weatherbee et al 1999).. Das derzeitige Protokoll hat sich von einer ausführlichen schriftlichen Protokoll auf Anfrage von der Carroll Lab für die Larven Flügel Färbungen angepasst. Die Veränderungen, die wir gemacht dienen, um Unterschiede zwischen Larven-und Puppenstadium Flügel Gewebe aufnehmen. Während Puppenstadium Hi...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Fix Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4% Paraformaldehyde in PBS</td>
</tr>
<tr>
<td>PBT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.1% Tween 20 in PBS</td>
</tr>
<tr>
<td>Proteinase K solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2.5&#xF06D;g/ml Proteinase K in PBT</td>
</tr>
<tr>
<td>Digestion Stop Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2 mg/ml glycine in PBT</td>
</tr>
<tr>
<td>Pre-Hybridization Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For 50 ml PHB: 12 ml DEPC treated water + 25 ml Formamide + 12.5 ml 20 x SSC + 50 ul Tween 20 + 500 ul 10 mg/ml salmon sperm (Rnase free, heat denatured prior to addition to solution).
</td>
</tr>
<tr>
<td>Hybridization Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Add 1 mg/ml glycogen to prehybridization buffer</td>
</tr>
<tr>
<td>Block Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50 mM Tris pH 6.8, 150 mM NaCl, 0.5% IGEPAL (NP40), 5 mg/ml BSA
</td>
</tr>
<tr>
<td>Anti-DIG</td>
<td>Ab</td>
<td>Roche Applied Science</td>
<td>11 093 274 910</td>
<td>Alkaline Phosphatase conjugated</td>
</tr>
<tr>
<td>DIG Wash and Block Buffer Set</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11 585 762 001</td>
<td>&nbsp;</td>
<tr>
<td>Crystal Mount Aqueous Mounting Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C0612</td>
<td>Mounting Medium </td>
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in situ protocol for butterfly pupal wings using riboprobes

Hier präsentieren wir Ihnen im Video-Format, ein Protokoll für in situ Hybridisierungen in Puppenstadium Flügel des Schmetterlings Bicyclus anynana mit Ribosonden. In situ Hybridisierungen, sind eine tragende Säule der Entwicklungsbiologie, nützlich, um die räumlichen und zeitlichen Muster der Genexpression in Geweben entwickeln auf der Ebene der Transkription zu studieren. Wenn Antikörper, die das Protein Produkte der Gen-Transkription Ziel noch nicht entwickelt worden, und / oder gibt es mehrere Genkopien eines bestimmten Proteins in das Genom, die nicht differenziert werden können mit den verfügbaren Antikörper ist, kann in situs verwendet werden. Während eine in-situ-Technik für die Larven Flügel-Discs verfügbar war, um den Schmetterling Gemeinde seit mehreren Jahren hat sich die aktuelle Protokoll für die größeren und fragile Puppenstadium Flügel optimiert.

Watch this Scientific Journal Video about In situ Protocol for Butterfly Pupal Wings Using Riboprobes at JoVE.com

In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Um die Genexpression in den Puppenstadium Flügel Gewebe Bicyclus anynana untersuchen, stellen wir ein optimiertes Protokoll für in situ Hybridisierungen mit Ribosonden. Wir bieten auch Richtlinien für die weitere Optimierung des Protokolls für den Einsatz in Puppenstadium Flügel der andere Lepidopteren Arten.

In situ-Protokoll für Butterfly Puppenstadium Flügel mit Ribosonden

Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes.  In situ ...

in-situ-protocol-for-butterfly-pupal-wings-using-riboprobes

Research

JoVE Journal

Biology

10.0K Aufrufe.  SUNY-University at Buffalo. Um die Genexpression in den Puppenstadium Flügel Gewebe Bicyclus anynana untersuchen, stellen wir ein optimiertes Protokoll für in situ Hybridisierungen mit Ribosonden. Wir bieten auch Richtlinien für die weitere Optimierung des Protokolls für den Einsatz in Puppenstadium Flügel der andere Lepidopteren Arten.

Serie: In situ Protocol for Butterfly Pupal Wings Using Riboprobes

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

Nicht radioaktiv In situ Hybridisierung Protocol Einsetzbar für Fichte und einer Reihe von Pflanzenarten

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Forschung

Biologie

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluoreszenz In situ Hybridisierung (FISH)-Protokoll in menschlichen Spermien

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Puppenstadium Abdomen Immunhistochemie

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Schnell Fluorescent<em&gt; In-situ-</em&gt; Hybridisierung Protokoll für Xist-RNA in Kombination mit Immunfluoreszenz von Histonmodifikationen in X-Chromosom-Inaktivierung

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissection und Montage<em&gt; Drosophila</em&gt; Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

Methoden zur Inszenierung Pupal Perioden und Messung der Flügel Pigmentierung der Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

Ein Protokoll für Labor Housing von Türkis Killifische (Nothobranchius Furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

Risikogefährdete Programme zur Vermehrung von Schmetterlingen, um das Wissen um die Lebensgeschichte und effektive Ex-Situ-Konservierungstechniken zu verbessern

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Probing mRNA Kinetics in Space and Time in Escherichia coli mit Zwei-Farben-Einmolekülfluoreszenz in Situ-Hybridisierung

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...