Sriharsa Pradhan, 섀넌 모리 키니, 잠시 만요 경인 친, Jurate Bitinaite, 유 청, 피에르 올리비에 Esteve, Romualdas Vaisvila, 스티븐 E. 야콥센의 연구소, UCLA. 이 작품은 부분적으로 NIH 1R44GM095209 - 01에 의해 지원되었다.

1. DNA의 Glucosylation 및 제어 반응 <ol><li> 얼음 1.5 밀리리터 반응 관에서 게놈 DNA의 50-10 마이크로 그램 (30 마이크로 그램 / 밀리리터의 최종 농도의 경우), UDP - 포도당의 12.4 microliters (80 micromolar의 최종 농도에 대한) NEBuffer 4 31 microliters를 섞어 및 최대 310 microliters nuclease - 무료 물 310 microliters로 전체 볼륨을 가지고 있습니다. </li><li> 155 microliters 각각 두 개의 튜브에이 반응 혼합물을 분리. </li><li> 어느 튜브에 T4 - β - glucosyltransferase 30 단위, 또는 3 microliters를 추가합니다. 부드럽게 최대 pipetting 아래로 잘 섞는다. 두 번째 관 제어 반응이기 때문에 대신 T4 - BGT의, 물 3 microliters를 추가합니다. </li><li> T4 - BGT이 예제에서 5 hydroxymethylcytosine 그룹의 수산기 그룹에 포도당을 추가되는 기간 동안 12 ~ 18 시간 동안 섭씨 37도에서 모두 튜브를 품어. </li></ol> 2. 제한 효소 분해 <ol><li> 레이블 세 0.2 밀리리터 PCR - 스트립 튜브 번호 3 1. 각각에 반응 혼합물의 나누어지는 50 UL. </li><li> 레이블 세 0.2 밀리리터 PCR - 스트립 튜브 번호 4-6. 각각으로 제어 혼합물의 나누어지는 50 UL. </li><li> 튜브 1 사에 MspI의 100 단위 또는 1 microliter를 추가합니다. 부드럽게 최대 pipetting 아래로 잘 섞는다. </li><li> 튜브 2와 5로 HpaII 50 단위 또는 1 microliter를 추가합니다. 부드럽게 최대 pipetting 아래로 잘 섞는다. </li><li> 그들이 통제하는 한 튜브 3 6 아무것도 추가하지 마십시오. </li><li> 최소한 4 시간 동안 섭씨 37도에서 모두 6 튜브를 품어. </li><li> 각각의 튜브에 Proteinase K 1 microliter를 추가하고 30 분 섭씨 40도에서 알을 품다. </li><li> 10 분 섭씨 95 도에 잠복기로 Proteinase K를 Inactivate. </li></ol> 3. PCR / qPCR에 의해 DNA를 분석 이 프로토콜은 잘 수행하기 위해 표시되었습니다 엔드 포인트 PCR을위한 코의 LongAmp DNA 형성 촉매를 사용합니다. 기타 PCR 프로토콜을 대체하실 수 있습니다. <ol><li> 배 LongAmp DNA 형성 촉매 반응 버퍼, 10 millimolar dNTPs, 10 micromolar 앞으로 프리머 1 microliter, 10 micromolar 리버스 프라이머 1 microliter, 템플릿 3 microliters 1.5 microliters 10 microliters : 얼음 0.2 밀리리터 PCR 반응 관에 다음과 같은 구성 요소를 추가합니다 이전 단계 LongAmp DNA 형성 촉매의 DNA 중합 효소 1 microliter, 그리고 nuclease없는 물을 최대 50 microliters부터 DNA. 이러한 볼륨이 사용되는 PCR 프로토콜에 따라 변경될 수 있습니다. </li><li> 부드럽게 반응을 섞는다. 필요한 경우, 빠른 스핀에 의해 튜브의 바닥에 모든 액체를 수집합니다. </li><li> 온수 뚜껑없이 thermocycler를 사용하는 경우에는 미네랄 오일 샘플을 중첩. </li><li> 섭씨 94 도까지 preheated 블록과 얼음 thermocycler로 PCR 튜브를 전송하고 자전거 프로그램을 시작합니다. 일상적인 3 단계 PCR의 경우, 15 초 동안 변성 섭씨 94도 30주기, 30 초 동안 풀림 섭씨 55-65도, 및 확장 섭씨 65도 다음에 30 초 동안 섭씨 94 도로 한 초기 변성 단계 있어야 20초 또는 킬로바이트 50 초. 이것은 5 분 섭씨 65 도에 마지막으로 확장하여 다음해야합니다. 실시간 PCR은 또한 5 methylcytosine 5 - hydroxymethylcytosine의 금액을 quantitate을 수행할 수 있습니다. 구체적인 내용을 neb.com에있는 제품 매뉴얼을 참조하십시오. </li></ol> 4. 대표 결과 : <img src="/files/ftp_upload/2661/2661fig1.jpg" alt="그림 1" /> 그림 1. 다른 마우스 Balb / C 조직 샘플에서 로커스 12 5 hydroxymethylcytosine 금액의 비교. (A), 엔드 포인트 PCR. (B), 실시간 PCR. 네 마우스 조직에서 DNA 분석했다. 비교 목적을 위해 실시간 PCR 데이터를 삭제하지 않은 DNA에 정상화되었다. 표준 곡선은 사본 번호를 결정하는 데 사용되었다. 샘플은 소화되지 않은있는 컨트롤의 복사본 번호 (없음 5)에 의해 시료 없음 1-6의 사본 번호를 나누어 정상화 수 있습니다. 박스 겔 레인 5 HMC 선물의 편차를 보여줍니다.

<ol>
	<li>Sadri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acids Res. 24, 4987-4989 (1996).</li><li>Mathur, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gene. 108, 1-6 (1991).</li><li>Butkus, V., Petrauskiene, L., Maneliene, Z., Klimasaukas, S., Laucys, V., Janulaitis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic Acids Res. 15, 7091-7102 (1987).</li><li>Kriaucionis, S., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 324, 929-930 (2009).</li><li>Tahiliani, M., Koh, K. P., Shen, Y., Pastor, W. A., Bandukwala, H., Brudno, Y., Agarwal, S., Lyer, L. M., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 324, 930-935 (2009).</li><li>Huang, Y., Pastor, W. A., Shen, Y., Tahiliani, M., Liu, D. R., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PLoS One. 5 (1), e8888-e8888 (2010).</li></ol>

테드와 Romas 모두는이 문서의 저자는 코의 직원되며 Romas 테드이 키트의 개발을 감독 응용 프로그램 및 개발 부서의 이사 있기 때문에 키트의 주요 개발자입니다.

본 실험을 설정할 때 고려해야 할 몇 가지 중요한 것들이 있습니다. 첫째, 게놈 DNA의 glucosylation 끝까지 진행하는 것이 중요합니다. T4 - BGT의 시퀀스 특이성이 알려져 있으며 그것은 기판 시퀀스에 대한 선택의 여지가 없어 보인다되지 않습니다. 따라서, 어떤 경우에, 더 이상 부화 시간이 필요할 수 있습니다. 둘째, MspI 및 HpaII 소화 배경 신호를 방지하기 위해 완료해야합니다. 이러한 제한 효소의 경우, 우리는 4 시간 배양 시간을 권장하지만, 불완전 절단이 관찰되면 더 이상 incubations을 수행할 수 있습니다. 셋째, 입력 DNA 금액은 DNA의 20 ngs는 엔드 포인트 PCR에 사용할 수 있기 때문에, 상황에 따라 조정할 수 있습니다. 마지막으로, 우리는 5 - MC 5 - HMC의 부량에 대한 게놈 DNA와 병렬로 실행할 수 있습니다 공급 제어 DNA의 사용을 권장합니다.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>EpiMark&trade; 5-hmC and 5-mC Analysis Kit</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>E3317</td>
<td>&nbsp;</td>
<tr>
<td>Locus specific primers, flanking a CCGG site of interest</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Custom per experiment; provided by user</td>
</tr>
<tr>
<td>LongAmp&reg; Taq DNA Polymerase</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>M0323</td>
<td>&nbsp;</td>
<tr>
<td>dNTPs</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>N0447</td>
<td>&nbsp;</td>
<tr>
<td>molecular biology grade water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>		</tbody>
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high sensitivity 5-hydroxymethylcytosine detection in balb/c brain tissue

DNA의 hydroxymethylation은 DNA의 긴 알려진 수정이지만, 최근 epigenetic 연구의 초점이되었다. 포유 동물 DNA는 효소 predominately CPG dinucleotides의 맥락에서, 5 MC로 사이 토신 (C) 잔류물의 5 번째 탄소 위치에 수정됩니다. 5 MC 개발과 질병에 관련된 것으로 아르 효소의 Tet 가족에 의해 5 HMC에 효소 산화 의무가 있습니다. 현재, 5 - HMC의 생물 학적 역할은 완전히 이해되지 않지만 biomarker로서의 가능성 때문에 많은 관심을 생성합니다. 이것은 마우스 배아 줄기 (ES)와의 연결을 세포에서 5 hydroxymethylcytosine을 식별하는 몇 가지 획기적인 연구로 인해 발생합니다. bisulfite 시퀀싱 방법을 포함한 연구 기술, 쉽게 5 MC 5 - HMC를 구분할 수 없습니다. 몇 가지 프로토콜은 질량 분광법 분석이나 게놈 DNA의 소화 단일 nucleosides의 얇은 층 크로마 토그래피 액체 크로마 토그래피와 결합을 포함한 게놈의 5 hydroxymethylcytosine의 글로벌 양을 측정할 수 존재한다. 도트 얼룩 분석, immunofluorescence, 또는 hydroxymethylated DNA의 침전에 사용될 수 있지만, 이러한 항체가 하나의 기본 resolution.In 추가이없는 대상 5 hydroxymethylcytosine도 존재한다는 항체, 해상도는 immunoprecipitated DNA의 크기와에 대한 의존 microarray 실험이 프로브 디자인에 따라 달라집니다. 그것이 5 hydroxymethylcytosine은 epigenetic 규제의 게놈 또는 역할 존재 정확한 위치를 알 수 있기 때문에, 새로운 기술은 특정 로커스 hydroxymethylation을 식별할 수있는 필요합니다. EpiMark 5 HMC 5 - MC 분석 키트는 특정 loci에서 두 수정 구별을위한 솔루션을 제공합니다. EpiMark 5 HMC 5 - MC 분석 키트 5 methylcytosine 및 특정 DNA를 로커스 내에서 5 - hydroxymethylcytosine의 식별 및 quantitation를위한 간단하고 강력한 방법입니다. 이 효소 접근 방식은 간단한 3 단계 절차에서 isoschizomers MspI과 HpaII의 차동 methylation 감도를 사용합니다. 관심 게놈 DNA는 5 hydroxymethylcytosine에 포도당 moeity를 추가 T4 - BGT와 함께 처리됩니다. 이 반응은 순서대로 독립적인, 따라서 모든 5 HMC이 glucosylated됩니다; 수정되지 않은 또는 5 - MC 포함된 DNA가 영향을받지 않습니다. 이 glucosylation는 다음 제한 endonuclease는 소화옵니다. MspI 및 HpaII 같은 순서 (CCGG)를 인식하지만 다른 methylation 상태에 민감합니다. 사이 토신 차단 절단 중 언제든지 변경 (5 - MC, 5 - HMC 또는 5 GHMC) : HpaII은 완전히 수정되지 않은 사이트를 클리브스. MspI 5 - MC 5 - HMC 아니지만 5 GHMC를 인식하고 클리브스. 프로토콜의 세 번째 부분은 PCR에 의한 로커스의 심문이다. 입력 DNA의 조금은 20 NG를 사용할 수 있습니다. 실험 (glucosylated와 소화) 및 제어 (모의은 glucosylated와 소화) 관심 CCGG 사이트 (100-200 BP)를 측면 primers와 대상의 DNA 증폭이 수행됩니다. CPG 사이트 5 hydroxymethylcytosine 포함되어있는 경우 밴드가 glucosylation과 소화 후에 감지 아니지만 비 glucosylated 제어 반응 인치입니다 실시간 PCR은 hydroxymethylcytosine이 특정 사이트에 얼마나의 근사치를 제공합니다. 본 실험에서는, 우리는 엔드 포인트 PCR에 의한 마우스 Babl / C 뇌 샘플에서 5 hydroxymethylcytosine 금액을 분석합니다.

Watch this Scientific Journal Video about High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue at JoVE.com

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

EpiMark 5 HMC 5 - MC 분석 키트는 분석하고 spe cific 로커스 이내 5 methylcytosine 5 - hydroxymethylcytosine을 quantitate하는 데 사용할 수 있습니다. 키트는 β - glucosyltransferase (T4 - BGT)를 이용한 효소 반응을 통해 5 - HMC의 수산기 그룹에 포도당의 추가에 의해 5 - HMC에서 5 - MC를 구별. 5 HMC는 CCGG의 맥락에서 발생하면,이 수정은 아닌 cleavable 사이트에 cleavable MspI 사이트를 변환합니다.

Balb / C 뇌 조직에 높은 민감도는 5 hydroxymethylcytosine 감지

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

high-sensitivity-5-hydroxymethylcytosine-detection-balbc-brain

Research

JoVE Journal

Neuroscience

14.9K 조회수.  New England Biolabs. EpiMark 5 HMC 5 - MC 분석 키트는 분석하고 spe cific 로커스 이내 5 methylcytosine 5 - hydroxymethylcytosine을 quantitate하는 데 사용할 수 있습니다. 키트는 β - glucosyltransferase (T4 - BGT)를 이용한 효소 반응을 통해 5 - HMC의 수산기 그룹에 포도당의 추가에 의해 5 - HMC에서 5 - MC를 구별. 5 HMC는 CCGG의 맥락에서 발생하면,이 수정은 아닌 cleavable 사이트에 cleavable MspI 사이트를 변환합니...

비디오: High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Immunoprecipitation 및 아실-비오틴 거래소 (아베)에 의한 양식 Hippocampal 뉴런의 단백질 Palmitoylation의 감지

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

연구

신경과학

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

조직 추출물의 고해상도 핵 자기 공명 분광법에 의한 쥐 뇌의 대사 체 분석

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

쥐 뇌 조직 검사 정렬 형광 활성 셀을 사용하여 셀 타입 별 유전자 발현

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

마우스에 콜드 민감도 및 적응을 결정하기위한 간단하고 저렴한 방법

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

고해상도를 사용하여 뇌 섹션에서 Axonally 지역화 된 mRNA를 검출<em&gt; 현장에서</em&gt; 하이브리드

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

MALDI 이미징 질량 분석에 대한 쥐 뇌 조직에 강글리오사이드의 감지 및 시각화를위한 DAN 매트릭스의 승화

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Drosophila 섬 분석 결과에서 운전 행동의 높은 처리량 분석

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

용매 클리어 뇌 조직에서 광견병 바이러스 감염의 고해상도 3D 이미징

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

근 적외선 형광 및 고 분해능 주사를 사용 하 여 설치류 뇌의 단백질 발현 측정

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

신경 줄기 세포와 쥐의 뇌에서 5-하이드록시메틸시토신의 검출

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...