Tinción de secciones de parafina <ol><li> Dewax diapositivas con xileno (Fisher X3 S -4) 3 veces durante 5 minutos cada uno (xileno cambian cada mes dependiendo del uso, xileno es tóxico). Use platos y tapas de 20 diapositivas de Fisher ref 08-812-1A y bastidores de hecho a la medida de estos platos. 200 ml de líquido por cada plato. </li><li> Hidratar a través del gradiente de alcohol de la siguiente manera (el cambio de gradiente cada 2 semanas): <ul><li> 2x en el 100% de etanol (Fisher # A406-20) durante 2 minutos cada uno </li><li> 2x en el 95% de etanol durante 2 minutos cada uno </li><li> 1x en el 70% de etanol durante 2 min </li><li> 1x en el 50% de etanol durante 2 min </li><li> 1x en el 30% de etanol durante 2 min </li><li> 1x ddH2O durante 2 minutos </li></ul></li><li> Remoje en DPBS durante 5 minutos (DPBS = fosfato Dulbecco modificado solución salina tamponada con Ca 2 + y Mg 2 +). </li></ol> Antígeno de recuperación: <ol><li> Pre-caliente el Na-citrato buffer (10 mM, pH 6,5) en el microondas. </li><li> Cocine las diapositivas durante 15 minutos en el microondas. Las diapositivas no debe dejarse secar a fin de comprobar cada minuto y añadir Na-citrato cuando sea necesario. </li><li> Deje que se enfríe a temperatura ambiente. </li><li> Bloquear la actividad de la peroxidasa endógena con 1% de H 2 O 2 en DPBS durante 20 minutos (1,5 ml de 30% de acciones Sigma H-1009 en 50 DPBS ml). </li><li> Remoje en DPBS 3 x 5 min. </li><li> Bloque no específicas sitios mediante la incubación de la noche a 4 ° C en DPBS + 5% de albúmina sérica bovina (BSA) + 0,1% de azida Na + 5% de suero. Nota: La elección de suero dependerá de las especies en que los anticuerpos utilizados para la tinción se realizaron. Bloque con el suero de la especie en la que el Ab secundario (conjugado con biotina) se hizo. Si esto no está disponible, el uso de suero de una especie diferente de aquel en el que se realizó el Ab primaria. </li><li> Bloquear los sitios de la biotina con el kit de avidina / biotina bloqueo (Vector laboratorios Catálogo # SP-2001): <ul><li> Incubar con avidina D durante 15 minutos. Aclarar brevemente con DPBS. </li><li> Incubar con la solución de biotina durante 15 minutos. </li></ul></li><li> Incubar en primaria Ab durante 2 horas a temperatura ambiente, en DPBS + 2% de BSA NaAzide + 0,1% + 2% de suero (igual que para bloquear el paso). </li><li> Remoje en PBS 3 x 5 min. </li><li> Incubar con el Ab secundario conjugado con biotina durante 1 hora a temperatura ambiente en DPBS + 2% de BSA + 0,1% de azida Na + 2% de suero (la misma que utiliza para el paso de bloqueo). </li><li> Prepare el reactivo ABC, al mismo tiempo, ya que tiene que desarrollar por lo menos 30 minutos antes de su uso! (Ver Materiales) </li><li> Preparar en un tubo cónico de 50 ml. En 15 ml de DPBS (sin suero, sin azida) añadir 3 gotas de reactivo A, mezclar y agregar 3 gotas de reactivo B y mezclar. Evite apretar las botellas, y no esperar a que las gotas para formar por su cuenta. ABC kit Vectastain PK-6100 de los laboratorios de Vector. </li><li> Remoje en DPBS 3 x 5 min. </li><li> Incubar con reactivo ABC durante 30-45 minutos a temperatura ambiente. </li><li> Remoje en DPBS 3 x 5 min. </li><li> Prepare el sustrato DAB peroxidasa (Vector Labs. # SK-4100) en 5 ml ddH2O en un frasco de vidrio inmediatamente antes de su uso (ver Materiales) <ul><li> 2 gotas de la solución de reserva de estabilización, la mezcla </li><li> 4 gotas de DAB, la mezcla (debe ser ligeramente marrón) </li><li> 2 gotas de H 2 O 2, la mezcla </li></ul></li><li> Caída del sustrato DAB en la parte superior de las diapositivas y ver para la tinción de color marrón. </li><li> Dip se desliza en el hielo + agua del grifo para detener la reacción. </li><li> Enjuague con agua fría del grifo durante 5 minutos. </li><li> De contraste con hematoxilina (20 seg; Fisher CS401-D) y enjuague con agua del grifo hasta que el agua salga clara. </li><li> Deshidratar el alcohol a través de gradiente a partir de etanol al 30% hasta el 100% de etanol (2 minutos cada uno). </li><li> Sumerja en xileno 3 x 5 min. </li><li> Montar con Permount (Fisher # SP15-100): poner un poco encima de la sección en la diapositiva y presione lentamente a la sección con un cubreobjetos sin burbujas de aire. No mueva el cubreobjetos hasta que esté completamente seco, lo cual lleva alrededor de 24 horas. </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

Inmunohistoquímica (IHC) es una técnica valiosa utilizada para localizar / visualizar la expresión de proteínas en una sección de tejido montado utilizando anticuerpos específicos. Hay dos métodos: el método directo e indirecto. En este experimento, sólo se describe el uso de la tinción inmunohistoquímica indirecta. Indirectos tinción IHC utiliza muy anticuerpos específicos de primaria y secundaria conjugado con biotina. Anticuerpos primarios se utilizan para identificar discretamente proteínas de interés mediante la unión a un epítopo específico, mientras que los anticuerpos secundarios reste para la tinción de fondo inespecífica y amplificar la señal mediante la formación de complejos con el anticuerpo primario. Las diapositivas pueden ser generados a partir de secciones congeladas, o secciones de parafina montados en portaobjetos de vidrio. En este protocolo, se discute la preparación de las secciones incluidas en parafina por desparafinación, hidratación usando un gradiente de alcohol, la recuperación de antígeno inductor de calor, y el bloqueo de la actividad peroxidasa endógena y no específicos de los sitios de unión. Algunas secciones se tiñen con anticuerpos específicos para el marcador de las células T CD8 y mientras que otras se tiñen de la tirosina hidroxilasa. Las diapositivas son posteriormente tratados con anticuerpos adecuados secundario conjugado con biotina, y luego desarrollado utilizando avidina conjugada con peroxidasa de rábano (HRP) con Diaminiobenzidine (DAB) como sustrato. Tras el desarrollo, las diapositivas se counterstained para el contraste, y se montaron en cubreobjetos con Permount. Después de un secado adecuado, estas diapositivas están listas para la imagen.

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

Inmunohistoquímica: las secciones de parafina utilizando la Vectastain ABC Kit de Vector Labs

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

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69.4K vistas.  University of California, Irvine (UCI). 

Video: 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.

Purificación del ADN plásmido de las colonias de bacterias Utilizando el kit de Qiagen Miniprep

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

Investigación

Biología

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.

Enriquecimiento de las células NK a partir de sangre humana con el Kit de RosetteSep de Tecnologías de la StemCell

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

La extracción de RNA a partir de células Neuroprecursor utilizando el kit de Bio-Rad ARN total

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.

Inmunohistoquímica en secciones de parafina de la epidermis del ratón Utilizando anticuerpos fluorescentes

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.

Las secciones incluidas en parafina y congeladas de Drosophila Los músculos de Adultos

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.

Extracción de ADN a partir de material incluido en parafina para análisis genéticos y epigenéticos

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.

Criopreservación de espermatozoides de ratón y de recuperación con el I · Kit de Cryo

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.

Generación de las células madre pluripotentes inducidas a partir de sangre periférica mediante el STEMCCA vector lentiviral

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.

La detección de formas modificadas de la citosina Uso Sensitive Inmunohistoquímica

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.

Un Protocolo Automatizado Rápido de Fibra Análisis Población en músculo de rata Secciones representativas Uso de la cadena pesada de miosina inmunohistoquímica

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