Este trabajo fue financiado por la Fundación Nacional de Ciencias naturales de China (Grant no. 31400936, 31460260) y la Ciencia Natural Fundación de Jiangxi provincia de China (20171BAB215020). También agradecemos al programa conjunto entre la Universidad de Nanchang y Queen Mary University of London para apoyar este trabajo.

Todos los métodos aquí descritos han sido aprobados por el cuidado Animal y el Comité de uso de la Universidad de Nanchang.
1. monta el equipo y conecte el microtomo
<ol><li>Diseño de los parámetros conforme a los requisitos (suplementario figura 1).</li>
	<li>Enviar el parámetro a una fábrica local para la fabricación de los tableros de acrílico.</li>
	<li>Montar todas las piezas en orden: utilizar cloroformo para combinar 7 comerciales tableros de acrílico en un tanque con un baño de agua y canal de sección (figura 1). PRECAUCIÓN: El cloroformo produce sustancias tóxicas cuando encuentra con luz y al oxígeno. Montar el aparato en una campana de humos.</li>
	<li>Instale el elemento de calefacción tubular a través del agujero en el tablero de acrílico #1 (punta de flecha de lafigura 2A, ).</li>
	<li>Para instalar el controlador de temperatura, crear un pequeño agujero en el tablero de acrílico #2 usando un taladro eléctrico e instale el detector de temperatura (flecha de lafigura 2A, ). Instalar un termostato digital en el tanque (figura 2A). Nota: El detector de temperatura debe estar dentro de 1 cm por debajo de la superficie del agua.</li>
	<li>Para proporcionar acceso a energía, ajustar la tensión inferior a 24 V antes de encender el equipo.</li>
	<li>Retire la bandeja de residuos de sección del microtomo.</li>
	<li>Enlace al canal de secciones con sujetador de la cuchilla de micrótomo con sellante neutral del silicón (figura 2B). Nota: No borrón de transferencia cualquier sellante neutral del silicón sobre la parte superior de la hoja de manera que la hoja vieja se sustituye fácilmente.</li>
	<li>Seco durante la noche para permitir que el cloroformo y el tiempo de sellador de silicona neutra para solidificar.</li>
	<li>Instalar la hoja en el portahoja. Vierta agua en el baño de agua (figura 2). La superficie del agua sólo debe tocar la parte superior de la hoja (Figura 2D, cabeza de flecha).</li>
</ol>2. parafina de seccionamiento
Nota: Desechos a menudo se acumulan en los bordes superiores o inferiores de la manzana de cera y la superficie del agua. Esta basura debe ser limpiado regularmente.
<ol><li>
Realizar la parafina convencional sección10.
	<ol>
<li>Fije a la muestra de parafina-encajado en un micrótomo rotatorio y coloque la cuchilla en un sujetador de la cuchilla.</li>
		<li>Gire el volante y la sección de la muestra.</li>
		<li>Transferencia de la cinta de parafina seccionado para el baño de agua caliente de 38,5 ° C.</li>
		<li>Pick up que las secciones en el microscopio se deslizarán de la superficie del agua después de desplegar.</li>
		<li>Seque el portaobjetos en un horno a 42 º C noche.</li>
	</ol>
</li>
	<li>
Secciones de parafina mejorada (figura 3)
	<ol>
<li>Encender el equipo.</li>
		<li>Abra el calentador con interruptor de detección de temperatura y temperatura específicas entre 38,0 ° C y 40,0 ° C durante 1 hora con antelación calentar el agua.</li>
		<li>Modificar el bloque de parafina adquirido por inclusión en parafina; luego coloque el bloque de parafina en abrazadera del cassette del microtomo y bloquéela. Nota: Los límites superiores e inferiores del bloque de parafina deben modificarse horizontalmente para evitar que la cinta de la sección de formando un arco y ser capaz de fluir a través del canal de secciones estrechas.</li>
		<li>Acercarse rápidamente a la muestra por secciones gruesas de corte.</li>
		<li>Ajuste a un espesor de sección adecuada (10 μm para cerebro de ratón adulto, ratón adulto riñones y cerebro de ratón embrión, 4 μm para los ojos de pez cebra). Gire el volante y las secciones entrará automáticamente en el canal de las secciones y el baño.</li>
		<li>Sección de la muestra en un movimiento lento y uniforme del corte (Figura 3A). Nota: Thethickness del primer tramo se verá alterada tras el volante se ha detenido durante unos minutos. Las cuchillas desechables deben reemplazarse cuando las secciones tienen arañazos consecutivos.</li>
		<li>Guía de las secciones en la cámara de baño flotan en una línea (figura 3B, 3C). Nota: Si la parte de la cabeza se adhiere a la pared de la cámara, utilice a una guía cepillo para mantenerlo flotando.</li>
		<li>Se adhieren a las secciones sobre portaobjetos. Después de las secciones completamente se despliegan en el baño de agua, varias secciones con pinzas. Luego recogerlos desde el baño de agua sobre el portaobjetos (figura 3D). Nota: Secciones deben recogidas en la parte posterior de la bañera de agua, como los del canal de las secciones y la parte anterior de la bañera de agua no todavía completamente desplegados.</li>
		<li>Seque las diapositivas colocándolas en un horno de 42 ° C durante la noche. Las diapositivas entonces pueden ser almacenadas a temperatura ambiente indefinidamente.</li>
	</ol>
</li>
	<li>Utilizar la tinción de hematoxilina-eosina (HE) para la evaluación del método mejorado8.</li>
</ol>

<ol><li>Chung, K., Deisseroth, K. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/23722210">CLARITY for mapping the nervous system.</a> Nature Methods. 10 (6), 508-513 (2013).</li>
<li>Fujita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+neuron+differentiation+in+the+central+nervous+system+by+tritiated+thymidine+autoradiography%5BTitle%5D)+AND+%22Journal+of+Comparative+Neurology%22%5BJournal%5D)">Analysis of neuron differentiation in the central nervous system by tritiated thymidine autoradiography.</a> Journal of Comparative Neurology. 122 (3), 311-327 (1964).</li>
<li>Mironov, V., Boland, T., Trusk, T., Forgacs, G., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organ+printing%3A+Computer-aided+jet-based+3D+tissue+engineering%5BTitle%5D)+AND+%22Trends+in+Biotechnology%22%5BJournal%5D)">Organ printing: Computer-aided jet-based 3D tissue engineering.</a> Trends in Biotechnology. 21 (4), 157-161 (2003).</li>
<li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryosectioning+tissues%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Cryosectioning tissues.</a> Cold Spring Harbor Protocols. 3 (8), (2008).</li>
<li>Viebahn, C., Luttenberg, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+anti-roll+plate+as+a+remedy+for+the+ill-effects+of+electrical+charge+during+cryosectioning%5BTitle%5D)+AND+%22Journal+of+Histochemistry+and+Cytochemistry%22%5BJournal%5D)">A modified anti-roll plate as a remedy for the ill-effects of electrical charge during cryosectioning.</a> Journal of Histochemistry and Cytochemistry. 37 (7), 1157-1160 (1989).</li>
<li>Onozato, M. L., Hammond, S., Merren, M., Yagi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+a+completely+automated+tissue-sectioning+machine+for+paraffin+blocks%5BTitle%5D)+AND+%22Journal+of+Clinical+Pathology%22%5BJournal%5D)">Evaluation of a completely automated tissue-sectioning machine for paraffin blocks.</a> Journal of Clinical Pathology. 66 (2), 151-154 (2013).</li>
<li>Sabaliauskas, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+zebrafish+histology%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">High-throughput zebrafish histology.</a> Methods. 39 (3), 246-254 (2006).</li>
<li>Chen, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hybrid-Cut%3A+An+Improved+Sectioning+Method+for+Recalcitrant+Plant+Tissue+Samples%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.</a> Journal of Visualized Experiments. (117), e54754(2016).</li>
<li>Kucherenko, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paraffin-Embedded+and+Frozen+Sections+of+Drosophila+Adult+Muscles%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles.</a> Journal of Visualized Experiments. (46), e2438(2010).</li>
<li>Cornell, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paraffin+Embedding+and+Thin+Sectioning+of+Microbial+Colony+Biofilms+for+Microscopic+Analysis%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis.</a> Journal of Visualized Experiments. (133), e57196(2018).</li>
<li>Whiteland, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemical+detection+of+T-cell+subsets+and+other+leukocytes+in+paraffin-embedded+rat+and+mouse+tissues+with+monoclonal+antibodies%5BTitle%5D)+AND+%22Journal+of+Histochemistry+and+Cytochemistry%22%5BJournal%5D)">Immunohistochemical detection of T-cell subsets and other leukocytes in paraffin-embedded rat and mouse tissues with monoclonal antibodies.</a> Journal of Histochemistry and Cytochemistry. 43 (3), 313-320 (1995).</li>
<li>Tucker, D. K., Foley, J. F., Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sectioning+Mammary+Gland+Whole+Mounts+for+Lesion+Identification%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Sectioning Mammary Gland Whole Mounts for Lesion Identification.</a> Journal of Visualized Experiments. (125), e55796(2017).</li>
<li>Venegas-Pino, D. E., Banko, N., Khan, M. I., Shi, Y., Werstuck, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+Analysis+and+Characterization+of+Atherosclerotic+Lesions+in+the+Murine+Aortic+Sinus%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus.</a> Journal of Visualized Experiments. (82), e50933(2013).</li>
<li>Lau, S. K., Chu, P. G., Weiss, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD163%3A+A+specific+marker+of+macrophages+in+paraffin-embedded+tissue+samples%5BTitle%5D)+AND+%22American+Journal+of+Clinical+Pathology%22%5BJournal%5D)">CD163: A specific marker of macrophages in paraffin-embedded tissue samples.</a> American Journal of Clinical Pathology. 122 (5), 794-801 (2004).</li>
<li>Campbell-Thompson, M. L., Heiple, T., Montgomery, E., Zhang, L., Schneider, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staining+Protocols+for+Human+Pancreatic+Islets%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Staining Protocols for Human Pancreatic Islets.</a> Journal of Visualized Experiments. (63), e4068(2012).</li>
</ol>

Los autores tienen una patente de este dispositivo y no declaran a intereses en competencia.

Para mejorar la morfología de la sección de parafina y resolver el problema del tiempo perdido durante el seccionamiento de la parafina convencional, hemos creado una parafina mejor método que combina el corte y el despliegue de seccionamiento. Este método mejorado se basa en un equipo simple que consta de un canal de sección, un baño de agua y un calentador con un interruptor de detección de temperatura. La cinta de la sección entra en el baño de agua a través del canal de sección y se despliega automáticame...

Estudio morfológico es importante en la investigación biológica. Aunque la nueva tecnología ha permitido a los investigadores a observar sus objetivos directamente en el tejido entero u organismos1,2,3, cortar al espécimen de secciones delgadas, seguido por la coloración, sigue siendo el principal morfología de tejido único método fornot sino directamente en el tejido de la proteína. La microscopia ligera utiliza tres tipos de sección: parafina, congelado y SEMIFINOS. Aunque cryosectioning es común para la protección de la antigenicidad del tejido, y la preparacion es simple, la morfología de tejido retenido es pobre e inadecuado para fina seccionamiento de4,5. Secciones de la parafina es el método más frecuentemente utilizado para exhibir bien preservada morfología. Ya que las muestras deshidratadas totalmente incrustadas en la cera, los bloques de parafina pueden ser almacenados indefinidamente. Además, secciones de la parafina produce secciones delgadas que mejoran el acceso de la sonda biológica en experimentos adicionales y reducen la superposición de capas de células en la dirección Z.
Sin embargo, secciones de la parafina convencional es tedioso y exige habilidad del operador. Secciones de la parafina se someten a la fijación, deshidratación, inclusión, corte y flotante. Lo importante, transferencia de cintas de la sección de soporte de la cuchilla en el baño de agua es necesaria pero difícil para los operadores junior. Especialmente en aire seco, las cintas de la sección se tuerza debido a la electricidad estática y son difíciles de desplegar en la superficie de agua caliente. Para mejorar la sección de calidad, humedecer la superficie de tejidos expuestos entre pasadas de cuchilla microtomo, enfriamiento los bloques de cera por inmersión en agua con hielo o aumentar la humedad con un humidificador cerca el micrótomo se recomienda6,7 . Nuevos métodos para mejorar la parafina secciones incluyen híbrido inclusión en parafina, cryosectioning8y sección comercial transferencia sistema asistencia9. Aunque estos métodos mejoran parcialmente parafina secciones velocidad y calidad, hacen secciones mucho más engorroso, y sistemas de transferencia de la sección comercial son caros.
En este protocolo, demostramos cómo crear simple, barato y flexible equipo paso a paso, que puede conectarse al sujetador de la cuchilla de un micrótomo rotatorio. Este equipo está compuesto por un canal de sección, un baño de agua y un calentador con un interruptor de detección de temperatura. Después del corte, docenas de secciones de flujo en el canal de sección y entrar en el baño de agua directamente, así se despliega automáticamente. Esto mejora la eficiencia de parafina de seccionamiento y hace que esta tecnología sea más conveniente. Usando este método, más adultas secciones de hipocampo de ratón, las secciones de riñón de ratón adulto, embrionario 15,5 días de edad (E15.5) ratón secciones del cerebro y ojo de pez cebra adulto secciones fueron cosechadas en menos tiempo e intacta más morfológicamente. Este método puede utilizarse también para otras muestras de tejidos que requieren acelerado parafina secciones evitando la pérdida de distinción de la sección.

<table><tbody><tr><td>Incubator</td><td>Boekel Scientific</td><td>133000-2</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>64-17-5</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>1330-20-7</td><td></td></tr><tr><td>Paraplast</td><td>Leica</td><td>39601006</td><td></td></tr><tr><td>Heated Paraffin Embedding Module</td><td>Leica</td><td></td><td></td></tr><tr><td>Commercial acrylic board</td><td></td><td></td><td></td></tr><tr><td>Trichloromethane</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>67-66-3</td><td></td></tr><tr><td>Tubular electric heating element(12 V 200 W)</td><td></td><td></td><td></td></tr><tr><td>Temperature controller(12 V 120 W)</td><td>Mingsuo</td><td>XH-W3002</td><td></td></tr><tr><td>Rotary microtome&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>Neutral silicone sealant</td><td></td><td></td><td>Link the water channel with the microtome knife holder</td></tr><tr><td>Voltage transformer</td><td>Dearll</td><td>S-250-12</td><td></td></tr><tr><td>Disposable blade</td><td>Accu-Edge</td><td>4689</td><td></td></tr><tr><td>Hematoxylin</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Baso Diagnostics Inc.</td><td>BA-4025</td><td></td></tr><tr><td>Microslide</td><td>Sail Brand</td><td>7105</td><td></td></tr><tr><td>Neutral balsam</td><td>Sinopharm Chemical Reagent Co.,Lid</td><td>10004160</td><td></td></tr><tr><td>Coverslip&amp;nbsp;</td><td>Citoglas</td><td>10212424C</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Xilong Chemical</td><td>7647-01-0</td><td></td></tr><tr><td>Water bath for paraffin sections</td><td>Leica</td><td></td><td></td></tr><tr><td>HistoCore Arcadia C&amp;nbsp;- Cold Plate</td><td>Leica</td><td></td><td></td></tr><tr><td>paraffin repellent spray&amp;nbsp;</td><td>Thermo Scientific</td><td>9990420</td><td></td></tr></tbody></table>

the cutting and floating method for paraffin-embedded tissue for sectioning

Secciones de tejido embebido en parafina es ampliamente utilizado en histología y patología. Sin embargo, es tedioso. Para mejorar este método, varias empresas han ideado sistemas de transferencia de sección compleja con fluido agua. Para simplificar esta tecnología, hemos creado un método sencillo usando equipos caseros que combina el corte y flotando dentro de una cámara termostática simple; por lo tanto, las secciones automáticamente entrar en el baño de agua en la superficie del agua. El hipocampo de cerebro de ratón adulto, los riñones de ratón adulto, cerebros embrionarios de ratón y ojos de pez cebra adulto fueron cortados usando secciones de parafina convencional y el método presentado para la comparación. El análisis estadístico muestra que nuestro método mejorado había ahorrado tiempo y produce perfiles de calidad superiores. Además, parafina de seccionamiento de la muestra entera en poco tiempo es fácil para los operadores junior.

El método mejorado aumentó el número de secciones de la parafina intacto. Probamos este nuevo método en tejido de hipocampo de ratón adulto, los riñones de ratón adulto, cerebros embrionarios de ratón y ojos de pez cebra. Se añade agua al tanque, y la temperatura del agua se mantuvo entre 38,0 ° C y 40,0 ° C. Después de una serie preparación de las muestras de tejido, fueron seccionadas y comparados con seccionamiento convencional. El nuevo método evita la pérdida de secci?...

Watch this Scientific Journal Video about The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning at JoVE.com

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

El método flotante para tejido parafina-encajado de seccionamiento y corte

Aquí, presentamos un protocolo para mejorar la parafina secciones. Este método combina el corte y flotando con una cámara termostática simple para evitar el proceso de transferencia requerido por el método convencional. Como resultado, la eficacia y el número de secciones de la parafina intacto mejoraron grandemente.

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

the-cutting-floating-method-for-paraffin-embedded-tissue-for

Research

JoVE Journal

Neuroscience

18.3K vistas.  Nanchang University. Aquí, presentamos un protocolo para mejorar la parafina secciones. Este método combina el corte y flotando con una cámara termostática simple para evitar el proceso de transferencia requerido por el método convencional. Como resultado, la eficacia y el número de secciones de la parafina intacto mejoraron grandemente.

Video: The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

El corte delgado de los preparativos de círculo para inmunohistoquímica

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Investigación

Biología

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Fluorescence in-situ hybridization (FISH) is often required in combination with histopathology and molecular diagnostics for selection of therapy in personalized medicine. A novel non-cross-linking, formalin-free tissue fixative that allows high quality morphologic, molecular and FISH analyses from the same specimen by addition of a post-fixation step before FISH is presented.

Protocolo para HER2 de los pescados usando un fijador de tejido no enlazan a la Cruz, libre de formol para combinar las ventajas de la criopreservación y la fijación de formalina

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its ...

Investigación sobre el cáncer

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Inclusión en parafina y finas secciones de Biofilms microbianos Colonia para el análisis microscópico

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Inmunología e Infección

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Trasplante en la almohadilla del ratón de ovario grasa

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Medicina

Establishment and Analysis of Tumor Slice Explants As a Prerequisite for Diagnostic Testing

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the multicellular interactions of native tissue. Tissue&#160;slices immediately cut from freshly resected tumors preserve spatial aspects of intratumor heterogeneity (ITH), thus making them useful surrogates of in vivo biology. Careful optimization of tissue slice preparation and cultivation conditions is fundamental for the predictive diagnostic potential of tumor slice explants. In this regard, murine models are valuable, as these provide a consistent flow of tumor material to perform replicate and reproducible experiments. This protocol describes the culturing of murine lung tumor-derived tissue slices using a rotating incubation unit, a system that enables intermittent exposure of tissues to oxygen and nutrients. Our previous work showed that the use of rotating incubation units improves the viability of tissue compared to other culture methods, particularly floating slices and stagnant filter supports. Here, we further show that slice thickness influences the viability of cultured slices, suggesting that optimization of slice thickness should be done for different tissue types. Pronounced ITH in relevant oncogenic functions, such as signaling activities, stromal cell infiltration or expression of differentiation markers, necessitates evaluation of adjacent tissue slices for the expression of markers altered by drug treatment or cultivation itself. In summary, this protocol describes the generation of murine lung tumor slices and their culture on a rotating incubation unit and demonstrates how slices should be systematically analyzed for the expression of heterogeneous tissue markers, as a prerequisite prior to drug response studies.

We provide a method for the generation, cultivation and systematic analysis of organotypic slices derived from murine lung tumors. We also describe how to optimize for slice thickness, and how to select drug concentrations to treat tumor slices.

Establecimiento y análisis de segmento de Tumor explantes como prerrequisito para la prueba de diagnóstico

Organotypic primary tissue explant cultures, which include precision-cut slices, represent the three-dimensional (3-D) tissue architecture as well as the ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Híbrido-Cut: un método de seccionamiento mejorada para muestras de tejido de la planta recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Islote pancreático inclusión de secciones de la parafina

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

<meta charset="utf8"></head><body>Modelo de ratón de endometrio fino optimizado con baja mortalidad y adherencias mínimas

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

<meta charset="utf8"></head><body>Establecimiento de un modelo de ratón de endometrio delgado

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Establecimiento de un modelo de ratón de endometrio delgado

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

El método flotante para tejido parafina-encajado de seccionamiento y corte

Resumen

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