パラフィン切片の染色<ol><li> 〜からろうを除去するが、各5分間3倍キシレン（フィッシャーX3 S -4）とスライド（用途に応じて毎月変更キシレン、キシレンは毒性があります）。これらの料理に合うように作られたフィッシャーREFから20のスライド08から812 - 1Aとラックの皿とカバーを使用してください。皿あたり200 mlの液体。 </li><li>次のようにアルコールの勾配を介して水和物（2週間毎に勾配を変更）​​： <ul><li> 2分ごとに100％エタノール（フィッシャー＃A406 - 20）で2倍</li><li> 2分ごとに95％エタノールで2倍</li><li> 2分間70％エタノールに1倍</li><li> 2分間50％エタノールに1倍</li><li> 2分間30％エタノールに1倍</li><li> 2分間1XのddH 2 O </li></ul></li><li> 5分（DPBS =ダルベッコ改変リン酸はCa 2 +およびMg 2 +との緩衝生理食塩水）のためのDPBSに浸す。 </li></ol>抗原の回復： <ol><li>電子レンジで予備加熱ナトリウム - クエン酸緩衝液（10mMの、pHは6.5）。 </li><li>電子レンジで15分間スライドを焼く。スライドは、すべての分をチェックし、Naのクエン酸、必要に応じて追加するように乾かしてはいけません。 </li><li>室温で冷ます。 </li><li> H 2は 20分（30％ストック50 mlのDPBSでシグマH - 1009を1.5 ml）のためのDPBSでO 2 1％で内因性ペルオキシダーゼ活性をブロックする。 </li><li> DPBS 3 × 5分に浸る。 </li><li> +4℃DPBSでC + 5％ウシ血清アルブミン（BSA）+ 0.1％のNaアジド+ 5％血清で一晩インキュベートすることにより、非特異的部位をブロックする。 注：血清の選択は、染色に使用される抗体が行われた種に依存します。二次抗体は、（ビオチンに結合体化）が行われた種からの血清でブロック。これが利用できない場合は、一次抗体が行われたものとは異なる種からの血清使用。 </li><li>アビジン/ビオチンブロッキングキット（ベクターラボラトリーズカタログ＃SP - 2001）とビオチンのサイトをブロックする： <ul><li> 15分間アビジンDでインキュベートする。 DPBSで簡単にすすいでください。 </li><li> 15分間ビオチン溶液でインキュベートする。 </li></ul></li><li> DPBSで室温で2時間、+ 2％BSA + 0.1％NaAzide +血清2％（ステップをブロッキングと同様）のために一次抗体でインキュベートする。 </li><li> PBSで3 × 5分に浸る。 </li><li> DPBSで室温で1時間+ 2％にビオチンを結合させた二次抗体とアジ化ナトリウムBSA + 0.1％+血清2％（ブロッキングのステップのために使われるのと同じ）インキュベートする。 </li><li>それは使用前に少なくとも30分間を開発する必要があるため、同時にABC試薬を準備します！ （資料を参照してください） </li><li> 50 mlコニカルチューブに準備します。 15mlのDPBS（無血清、無アジ化）で試薬を3滴、ミックスを追加し、試薬Bとミックスの3滴を追加します。ボトルを強く握ったりしないで、むしろ自分自身で形成するためにドロップを待ちます。 ABCキットステインPK - 6100ベクトルLabsから。 </li><li> DPBS 3 × 5分に浸る。 </li><li>室温で30-45分のためのABC試薬でインキュベートする。 </li><li> DPBS 3 × 5分に浸る。 </li><li> 使用直前にガラスバイアルに5ミリリットルのddH 2 OにおけるDABのペルオキシダーゼ基質（ベクターラボ＃SK - 4100）を準備する（資料を参照してください） <ul><li>バッファーのストック溶液を2滴、ミックス</li><li> DAB、ミックスの4滴（少し茶色になるはず） </li><li> 2滴H 2 O 2、ミックス</li></ul></li><li>茶色の染色用スライド、時計の上にDAB基質をドロップします。 </li><li>氷にディップスライド+反応を停止させる水をタップ。 </li><li> 5分間冷たい水道水ですすいでください。 </li><li>ヘマトキシリン​​（20秒、フィッシャーCS401 - D）との対比と水が出て明らかになるまで水道水ですすいでください。 </li><li>最大100％エタノール（2分ごと）に30％エタノールで出発アルコールの勾配によって脱水。 </li><li>キシレン3 × 5分に浸る。 </li><li> Permountとマウント（フィッシャー＃SP15 - 100）：いくつかのスライド上にセクションの上に置くと、気泡のないカバーとセクションの上にゆっくりと押してください。 〜24時間かかりますれ、完全に乾燥するまで、カバースリップを移動しないでください。 </li></ol>

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		<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>Xylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>X3S-4</td>
<td>Change xylene every month depending on use. Xylene is TOXIC.</td>
</tr>
<tr>
<td>100% ethanol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A406-20</td>
<td>Use to make EtOH gradient.</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dulbecco&#x2019;s modified Phosphate Buffered Saline WITH Ca2+ and Mg2+</td>
</tr>
<tr>
<td>Na-Citrate buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM, pH 6.5</td>
</tr>
<tr>
<td>H2O2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-1009</td>
<td>30% stock => Dilute to 1% in DPBS</td>
</tr>
<tr>
<td>Blocking solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>DPBS + 5% bovine serum albumin (BSA) + 0.1% Na Azide + 5% serum. </td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Azide</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>NaAzide</td>
</tr>
<tr>
<td>avidin/biotin blocking kit</td>
<td>&nbsp;</td>
<td>Vector laboratories</td>
<td>SP-2001</td>
<td>&nbsp;</td>
<tr>
<td>Ab buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>DPBS + 2% BSA + 0.1% Na Azide + 2% serum (same as used for the blocking step)</td>
</tr>
<tr>
<td>ABC kit Vectastain PK-6100</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DAB peroxidase substrate</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>SK-4100</td>
<td>&nbsp;</td>
<tr>
<td>ddH2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hematoxylin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>CS401-D</td>
<td>counterstain</td>
</tr>
<tr>
<td>Permount</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SP15-100</td>
<td>&nbsp;</td>
<tr>
<td>Slides and cover-slips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>glass</td>
</tr>		</tbody>
		</table>
		</div>

immunohistochemistry: paraffin sections using the vectastain abc kit from vector labs

免疫組織化学（IHC）は、ローカライズ/特異的な抗体を使用して、マウントされた組織切片でのタンパク質発現を視覚化するために利用価値のある技術です。直接的および間接的な方法：2つの方法があります。この実験では、我々は唯一の間接的なIHC染色の使用について説明します。間接IHC染色は特異性の高いプライマリおよびビオチン標識二次抗体を利用しています。一次抗体は、離散的に二次抗体が非特異的なバックグラウンド染色のために減算し、一次抗体に複合体を形成することにより信号を増幅しながら、特定のエピトープに結合することにより目的のタンパク質を識別するために利用されています。スライドは、どちらの凍結切片から生成、またはパラフィン包埋切片をガラススライドにマウントすることができます。このプロトコルでは、我々は、内因性ペルオキシダーゼ活性と非特異的結合部位のアルコールの勾配を使用して脱ろう、水和、熱誘導抗原の検索、および遮断することにより、パラフィン包埋切片の準備について説明します。いくつかのセクションは、T細胞マーカーCD8特異的な抗体で染色されているものと、チロシン水酸化酵素のために染色されている間。スライドが続いてビオチンに結合した適切な二次抗体で処理し、次いで基質としてDiaminiobenzidine（DAB）とアビジン標識西洋ワサビペルオキシダーゼ（HRP）を利用して開発した。開発に続いて、スライドは、コントラストのために対比されている、とpermountでカバースリップの下にマウント。十分乾燥した後、これらのスライドは、イメージングのための準備が整いました。

Watch this Scientific Journal Video about Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs at JoVE.com

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

免疫組織化学：ベクターLabsからベクタステインABCキットを用いたパラフィン切片

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

immunohistochemistry-paraffin-sections-using-vectastain-abc-kit-from

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Biology

69.4K ビュー.  University of California, Irvine (UCI). 

ビデオ: Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

生物学

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

StemCell Technologies社からRosetteSepキットと人間の血液からNK細胞の濃縮

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

Bio - Rad社のTotal RNA Kitを使​​用してNeuroprecursor細胞からのRNA抽出

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

蛍光抗体を用いたマウス表皮のパラフィン切片で免疫組織化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

遺伝学的およびエピジェネティクス解析のためのパラフィン包埋材料からのDNA抽出

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

I ·クライオキットを使用してマウスの精子の凍結保存と回復

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

STEMCCAレンチウイルスベクターを用いた末梢血からのヒト人工多能性幹細胞の生成

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

高感度免疫組織化学を使用してシトシンの修飾された形態の検出

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

ミオシン重鎖免疫組織化学を使用して、ラット筋断面における筋線維人口分析のための迅速な自動化されたプロトコル

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...