우리는 제인 Selegue, 마가렛 홀링스워스, 진 베리, 료 Futahashi, Najmus Sahar Mahfooz, 알렉산더 Popadic, 밥 리드,이 프로토콜을 문제 해결에 대한 도움을 로체 기술 지원을 주셔서 감사합니다. 우리는 또한 동영상을 편집에 대한 조언 윌리엄 피엘 감사합니다.

1 일 <ol><li> 다음과 같은 솔루션을 준비 : <ul><li> 10X PBS </li><li> 1X PBS </li><li> 1X PBT </li><li> ddH 2 O <ul><li> 0.1 % 총 볼륨의 DEPC를 추가하고 적극적으로 해결책을 흔들. </li><li> 압력솥. </li></ul></li><li> 4 % Paraformaldehyde 픽스 - PBS - DEPC를 사용하여 </li><li> Proteinase K 솔루션 - 침전물이 존재하는 경우, 소용돌이 적극적으로 나누어지는를 제거하기 전에 </li><li> 소화 스톱 버퍼 </li><li> Prehybridization 버퍼 </li><li> 50:50 PBT : Prehybridization 버퍼 </li><li> 하이브 리다이 제이션 버퍼 <ul><li> 솔루션은 RNase 무료 보관해야합니다. 어느 구운되었습니다 유리 도자기 (250에서 4시간 ° C) 또는 일회용 플라스틱을 사용합니다. Serological pipettes 솔루션을 만들기위한 매우 유용합니다 </li></ul></li></ul></li><li> PBS에서 가장 날개를 해부하다 시간 연출 pupae </li><li> 상온에서 24 자 문화 판의 우물에서 버퍼를 해결하려면 직접 날개 이동 </li><li> 2 시간 동안 디스크를 수정 </li><li> PBT 5 X 5 분 와시 </li><li> Proteinase K 용액에서 3 분간 날개를 품어 </li><li> 소화 스톱 버퍼 2 X를 린스 </li><li> PBT 5 X 5 분 와시 </li><li> 50:50 PBT 2 X 5 분 와시 : PHB </li><li> PHB 1 X 10 분 와시 </li><li> 55 PHB 적어도 1 시간에 품어 ° C. </li><li> 열 변성 RNA 프로브 (20 - 50ng 잘마다 필요한) (80 ° C 5 분)와 하이브리드화 버퍼 (100 μl 아니라 당 필요)에 추가 </li><li> 우물에 프로브를 추가하고 55에서 48 시간 품어 ° C </li></ol> 3 일 <ol><li> 55 4 X 5 분 씻어 ° C PHB </li><li> 55 PHB에 밤새 품어 ° C </li></ol> DAY 4 <ol><li> 다음과 같은 솔루션을 준비 : <ul><li> 항체 블럭 버퍼 </li></ul></li><li> PHB : 50:50 PBT 1 X 5 분 와시 </li><li> PBT 4 X 5 분 와시 </li><li> 4 블럭 버퍼에서 1 시간 동안 품어 날개 ° C </li><li> 4 박 안티 파 항체의 1:2000 희석에 날개를 품어 ° C </li></ol> 5 일 <ol><li> 다음과 같은 솔루션을 준비 : <ul><li> 감지 버퍼 </li></ul><ul><li> 개발 솔루션 - 민감한 빛 (호일에 포장) </li></ul></li><li> 상온에서 PBT 3 X 5 분 와시 </li><li> PBT의 와시 7 X 오분 </li><li> 감지 버퍼에 날개를 두 번 씻어 </li><li> 솔루션 개발 1 ML에 날개를 개발 - 개발의 시간은 각각의 시간을 모니터링해야합니다 - 동일한 프로브 및 개발 솔루션을 사용하면 개발 시간도 변경하실 수 있습니다 - 일반적으로 하루에 간격을 증가 후 50~10분 후 확인합니다. 플레이트는 샘플을 검사하지 않을 경우 빛의 노출을 방지하기 위해 호일에 싸여 있어야합니다. </li><li> 개발 중지 버퍼 다섯 번 린스 </li><li> 날개를 탑재합니다. </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>

기존 프로토콜의 최적화 애벌레 날개 디스크에 현장 hybridizations 성공적으로 대의 coenia의 나비의 디스크를 사용하여 수행되었습니다 (캐롤 외 1994;. 키 외 1999;. 웨더비 외 1999가).. 현재 프로토콜은 애벌레 날개 stainings에 대한 캐롤 연구소에서 요청에 따라 사용할 수있는 세부적인 서면 프로토콜에서 맞게되었습니다. 우리가 만든 변경 사항은 애벌레와 pupal 날개 조직 간의 차이를 수용할 수 있도록 제공하고 ...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

in situ protocol for butterfly pupal wings using riboprobes

여기서 우리는, 비디오 형식, riboprobes를 사용하여 나비 Bicyclus anynana의 pupal 날개의 현장 hybridizations에 대한 프로토콜을 제시한다. 현장 hybridizations에서 발달 생물학의 의지는 전사 수준에서 조직 개발에 유전자 표현의 공간 및 시간적 패턴을 연구하는 데 유용합니다. 유전자 전사의 단백질 제품을 대상 항체가 아직 개발 및 / 또는 사용 가능한 항체를 사용하여 차별화된 수 없습니다 게놈에서 특정 단백질의 여러 유전자 사본이있다하지 않은 경우, 시터스 대신 사용할 수 있습니다. 애벌레 날개 디스크 현장 기술에 몇 년 동안 나비 커뮤니티에 제공하고있는 동안, 현재의 프로토콜은 더 큰 더 연약한 pupal 날개에 대한 최적화되었습니다.

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

Bicyclus anynana의 pupal 날개 조직에서 유전자 발현을 조사하기 위해, 우리는 riboprobes를 사용하여 현장 hybridizations에 대한 최적화된 프로토콜을 제시한다. 우리는 또한 다른 Lepidopteran 종의 pupal 날개에 사용하기 위해이 프로토콜을 더욱 최적화에 대한 지침을 제공합니다.

Riboprobes를 사용하여 나비 Pupal 날개에 대한해라 프로토콜

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

9.9K 조회수.  SUNY-University at Buffalo. Bicyclus anynana의 pupal 날개 조직에서 유전자 발현을 조사하기 위해, 우리는 riboprobes를 사용하여 현장 hybridizations에 대한 최적화된 프로토콜을 제시한다. 우리는 또한 다른 Lepidopteran 종의 pupal 날개에 사용하기 위해이 프로토콜을 더욱 최적화에 대한 지침을 제공합니다.

비디오: 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.

비 - 방사성 현장에서 하이브 리다이 제이션 노르웨이 스프루스에 대한 해당 프로토콜과 식물 종의 범위

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

연구

생물학

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.

형광 현장에서</em인간의 정자에서> 하이브리드화 (물고기) 프로토콜

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 Pupal의 복부 Immunohistochemistry

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.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</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.

빠른 형광<em&gt; 현장에서</emX 염색체 불 활성화에 히스톤 수정의 면역 형광와 XIST RNA 결합에 대한&gt; 하이브리드 프로토콜

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.

해부와의 설치<em&gt; 초파리</em&gt; 번데기 아이 디스크

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.

준비 번데기 기간 및 초파리 guttifera 의 날개 색소 측정 방법

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.

실험실 주택의 청록색 송사리 (Nothobranchius furzeri)에 대 한 프로토콜

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.

생명역사 지식과 효과적인 Ex Situ 보존 기술을 향상시키기 위한 위험에 처한 나비 포로 전파 프로그램

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.

시투 혼성화에서 2색 단일 분자 형광을 사용하여 에샤리치아 대장균의 공간과 시간에서 mRNA 운동학을 프로빙

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