Este proyecto fue apoyado por la Fundación Nacional de Ciencias Naturales de China (SNCF): 31301950 y 31671288.

El método actual se ha establecido y validado para cosechar y almacenar más de 1.000 muestras de músculo para la tinción histológica en nuestro laboratorio. Todos los procedimientos que implican el cuidado y uso de animales a las directrices establecidas por el Ministerio de Agricultura de China. 1. Equipo para la recogida de muestras <ol><li> Etiquetar la identidad de la muestra en el vial criogénico. Nota: El vial criogénico está especialmente diseñado para congelar muestras de tejido fresco para el procedimiento de sección criogénica (Figura 1A). Los viales están hechas de polipropileno en un molde de fábrica (Figura 1C). </li><li> Obtener el siguiente equipo: nitrógeno líquido en un tanque de nitrógeno líquido apropiado, gafas de seguridad o un protector de cara, guantes congelador, 10 cm fórceps, 25 pinzas cm, un enfriador de espuma de poliestireno (dimensiones internas: 20 cm de largo x 12 cm de ancho x 13,5 cm de alto ; dimensiones exteriores: 24 cm de largo x 16 cm de ancho x 15 cm de altura), película de plástico (2,5 cm de largo x 0,8 cm de ancho x 0,1 mm de espesor),un bisturí, y las cubiertas de unión A4 PVC (21 cm x 29,5 cm) (Figura 1B). </li></ol> 2. Preparación de muestras de músculo <ol><li> Llenar el enfriador de poliestireno con nitrógeno líquido 5 min antes de la recogida de muestras. NOTA: El gran columna de nitrógeno líquido es esencial porque parte del nitrógeno líquido hierve lejos y se convierte en gas cuando las muestras frescas toquen. Con el fin de reducir la frecuencia de transferencia de nitrógeno líquido para reponer la pérdida, un enfriador de espuma de poliestireno de tamaño apropiado debe ser adquirida por adelantado de acuerdo con el número de muestras. </li><li> Una vez que se obtiene un espécimen, suavemente colocarlo en una pieza de la cubierta de unión A4 PVC y observar la dirección de las fibras musculares. NOTA: En este caso, hemos utilizado la sección transversal del músculo longissimus Doris desde la primera hasta la última vértebra lumbar en los cerdos. Los especímenes son de aproximadamente 12 cm de largo, 8 cm de ancho, y 5 cm de altura. </li><li> Utilizar un bisturí para realizar dos incisiones paralelas a través de la I Modelon la dirección de las fibras musculares, de aproximadamente 3 cm de longitud y 0,6 cm de distancia. Recorte el músculo incisa en un rectángulo de aproximadamente 0,6 cm de ancho x 0,6 cm de alto x 1,5 cm de largo. NOTA: La fiabilidad de la medición de la superficie de sección transversal de las fibras musculares depende principalmente de la orientación de las fibras del músculo incisa. </li><li> Utilice 10 fórceps cm para poner suavemente la muestra en un trozo de película de plástico, con el eje longitudinal del músculo paralelo incisa al borde largo de la película (Figura 2A). NOTA: La película de plástico tiene dos funciones: una función de sujeción que ayuda en la fijación de la orientación de las fibras musculares y una función de adherencia que impide que las grietas en la muestra debido a la rápida congelación. </li><li> El uso de pinzas, coloque el tejido muscular en el medio inferior de un vial criogénico de marcado, sólo entre los agujeros de entrada, y luego tapa fuertemente el vial (Figura 2B). </li></ol> 3. Congelación y Almacenamiento de muestra de músculos <ol><li> El uso de 25 pinzas cm, sumergir rápidamente todo el vial criogénico en nitrógeno líquido en el enfriador de poliestireno pre-enfriado, a la espera hasta que el nitrógeno líquido no hierva (aproximadamente 10-20 s). NOTA: El criovial flotará cuando el nitrógeno líquido está hirviendo, por lo sumerja cuidadosamente el vial en el nitrógeno líquido en ebullición durante 10-20 s. </li><li> La transferencia de las muestras de músculo a un tanque de almacenamiento de nitrógeno líquido o un congelador a -80ºC para almacenamiento a largo plazo. </li></ol> 4. Slide Preparación <ol><li> Preenfriar un criostato a entre -20 ° C y -22 ° C. </li><li> Retirar y desechar la vieja cuchilla del micrótomo, limpiar el soporte de la cuchilla y la placa estabilizadora en el criostato, instalar una nueva cuchilla del micrótomo en el criostato, y establecer el espesor de corte a 8 m. </li><li> Transferir la muestra del músculo en el vial criogénico del depósito de almacenamiento de nitrógeno líquido o -80 ° C congelador al criostato, su transporte en nitrógeno líquido o en hielo seco. </li&gt; <li> Esperar 30 min para el espécimen se equilibre con la temperatura del criostato. NOTA: Cualquier equipo que pueda tocar la muestra debe ser pre-enfría, porque una alta temperatura puede causar la formación de cristales de hielo en la muestra. </li><li> Montar la muestra en el soporte de la muestra bajo la formulación óptima temperatura de corte de glicoles y resinas solubles en agua (OCT). Cortar secciones 8-M. NOTA: Ajuste el ángulo de corte perpendicularmente a la orientación de las fibras musculares, debido a que el CSAF es el área de sección transversal de las fibras musculares. </li><li> Montar las secciones hacia el centro de un portaobjetos de microscopio a temperatura ambiente. Inmediatamente colocar el portaobjetos en una caja de portaobjetos en un congelador a -80ºC. No permita que el portaobjetos se seque a temperatura ambiente. </li></ol>

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No hay conflictos de interés, financiera o de otra manera, son declarados por los autores.

A continuación, describimos una nueva, criovial múltiples orificios para la congelación y el almacenamiento de los tejidos musculares para llevar a cabo las evaluaciones histológicas de la función muscular. El paso modificado crítico en este protocolo es que la muestra en el criovial multi-agujero se sumerge directamente en nitrógeno líquido. A nuestro entender, esta es la manera más simple y rapidest obtener excelentes muestras congeladas para cryosectioning muscular entre los métodos de congelación existent...

El músculo esquelético es el componente más valioso de un animal de producción de carne desde el punto de vista nutricional y procesamiento. En la industria de la carne, hay dos aspectos especialmente críticos: la eficiencia del crecimiento del músculo y la calidad de la carne resultante. Como componente principal del músculo, las fibras musculares están directamente relacionados con el crecimiento y la calidad de la carne fresca en animales 1. Por ejemplo, el número total de fibras (TNF) y el Área de sección transversal de fibras (CSAF) principalmente determinan la masa muscular y calidad de la carne; También, Tipo de fibra Composición (FTC) afecta en gran medida la calidad de la carne fresca 2. Por lo tanto, la manipulación de características de la fibra muscular en los animales es un método altamente eficaz para aumentar la rentabilidad del núcleo y competitividad de las explotaciones 1. Hasta la fecha, varios factores intrínsecos y extrínsecos se han identificado para manipular, ambie fibra muscularics 1. Esta manipulación se puede lograr a través de la selección específica de los animales con genes específicos, tales como el gen de la miostatina en el ganado 3, el gen Callipyge en ovejas 4, y el RYR1 y genes IGF2 en cerdos 5. Además, el control de la dieta y los tratamientos con hormonas específicas juegan un papel importante en características de la fibra muscular 6. Por lo tanto, un enfoque que combina los factores genéticos y nutricionales podría ser capaz de mejorar el contenido de carne magra y calidad de la carne. Sin embargo, los estudios sobre las fibras musculares están limitados en la industria de la carne debido a la elucidación de la estructura de las fibras musculares todavía es un reto. propiedades de la fibra muscular se identifican utilizando métodos histoquímicos, tales como el ensayo de la miosina adenosina trifosfatasa (ATPasa). Este método se basa en el hecho de que las enzimas encuentra en secciones delgadas (6-8 m) congelados defibras musculares se pueden hacer reaccionar químicamente con ciertos productos. Sin embargo, el contenido de agua de los músculos es mayor que 75% en cerdos, conejos, ratones y seres humanos, independientemente de la posición (es decir, la espalda, abdomen, o de las extremidades posteriores) 7. Tal alto contenido de humedad en los músculos causa un problema comúnmente encontrado - congelación artefactos - durante la preparación de criosecciones, como se describió previamente 8, 9. En la mayoría de los casos, es casi imposible para congelar adecuadamente los tejidos musculares en una línea de producción del matadero, de acuerdo con nuestra experiencia. El protocolo que se presenta aquí describe un método simple y eficiente utilizado en nuestro laboratorio para congelar los tejidos musculares para cryosectioning en una forma de alto rendimiento. El punto culminante de este método es un nuevo criovial que está diseñado para tejidos musculares de flash-congelación en nitrógeno líquido. El flujo de trabajo actual puede facilitar simultáneamente tejidocongelación y de procesamiento para una excelente cryosection muscular, con un compartimiento citoplasmático claramente visible y la estrecha aposición de miofibras en el tejido circundante. Además, este protocolo se puede aplicar a una amplia gama de opciones para el análisis del tejido porque el nitrógeno líquido no se mezcla con los tejidos.

<table border="0" cellpadding="0" cellspacing="0" width="508">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Cryostat Microtome</td>
			<td style="width:111px;">&#160;Leica</td>
			<td style="width:111px;">Leica CM1950</td>
			<td style="width:148px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Microscope&#160;</td>
			<td>Nikon</td>
			<td>Nikon DS-U3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">&#160;Cryogenic Vial&#160;&#160; Plastic film</td>
			<td></td>
			<td></td>
			<td>Designed by ourself</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cm-forcep</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25cm-tweezer</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Safety glass</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freezer gloves</td>
			<td></td>
			<td></td>
			<td>Commomly-used</td>
		</tr>
	</tbody>
</table>

a multi-hole cryovial eliminates freezing artifacts when muscle tissues are directly immersed in liquid nitrogen

Los estudios sobre la fisiología del músculo esquelético se enfrentan al desafío técnico de procesar adecuadamente las muestras para obtener secciones con compartimentos citoplasmáticos claramente visibles. Otro obstáculo es la aposición estrecha de las miofibrillas a los tejidos circundantes. Debido a que el proceso de fijación del tejido y la inclusión en parafina conduce a la contracción de las fibras musculares, de congelación es un medio óptimos de endurecimiento tejido muscular para seccionar. Sin embargo, un problema comúnmente encontrado, la formación de cristales de hielo, se produce durante la preparación de secciones congeladas debido a la alto contenido de agua de los músculos. El protocolo que se presenta aquí describe primero un método simple y eficiente para congelar adecuadamente tejidos musculares sumergiéndolos en nitrógeno líquido. El problema con el uso de nitrógeno líquido solo es que causa la formación de una barrera de gas nitrógeno al lado del tejido, que actúa como un aislante e inhibe la refrigeración de los tejidos. Para evitar este efecto &quot;manta de vapor&quot;, un necriovial w fue diseñado para aumentar la velocidad de flujo de líquido alrededor de la superficie del tejido. Esto se logró mediante la perforación de un total de 14 agujeros de entrada en la pared del vial. De acuerdo con la dinámica de burbujas, una mayor tasa de resultados de flujo de líquido en burbujas más pequeñas y menos posibilidades de formar una barrera a los gases. Cuando el nitrógeno líquido fluye en el criovial a través de los agujeros de entrada, la velocidad del flujo alrededor del tejido es lo suficientemente rápido para eliminar la barrera de gas. En comparación con el método de congelar los tejidos musculares usando isopentano previamente enfriado, este protocolo es más simple y más eficiente y se puede usar para congelar músculo de una manera rendimiento. Además, este método es óptimo para las instituciones que no tienen acceso a isopentano, que es extremadamente inflamable a temperatura ambiente.

La ilustración criovial y el equipo común de laboratorio para la congelación de los músculos durante la preparación de secciones congeladas se muestran en la Figura 1. Los crioviales están hechos de polipropileno en un molde de fábrica. Cada vial tiene un total de 14 agujeros de entrada: uno está en la tapa; otra es en la parte inferior; y los 12 forma de cuatro líneas paralelas restantes, cada uno con cuatro agujeros a 90º uno del otro. Estos orificios de entr...

Watch this Scientific Journal Video about A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen at JoVE.com

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Este protocolo describe un procedimiento para congelar los tejidos musculares al sumergirse directamente en nitrógeno líquido. Este protocolo también pone de relieve un nuevo criovial que puede evitar el &quot;efecto manta&quot; de gas nitrógeno cuando los contactos de nitrógeno líquido la superficie del tejido de un espécimen.

A Multi-agujero criovial Elimina congelación Artifacts cuando los tejidos musculares se sumergen directamente en nitrógeno líquido

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

a-multi-hole-cryovial-eliminates-freezing-artifacts-when-muscle

Research

JoVE Journal

Biology

11.2K vistas.  Jiangxi Agricultural University.  Este protocolo describe un procedimiento para congelar los tejidos musculares al sumergirse directamente en nitrógeno líquido. Este protocolo también pone de relieve un nuevo criovial que puede evitar el "efecto manta" de gas nitrógeno cuando los contactos de nitrógeno líquido la superficie del tejido de un espécimen. 

Video: A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Congelar las células madre embrionarias humanas

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Investigación

Biología

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Flash de congelación y muestras criostáticas E12.5 cerebro de ratón

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

Congelación, descongelación, y las Pilas de embalaje para el transporte

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Aip1p Dynamics Are Altered by the R256H Mutation in Actin

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of fluorescently tagged proteins using total internal fluorescence (TIRF) microscopy is used to visualize and quantify changes in cytoskeletal dynamics. TIRF microscopy and the use of fluorescent tags also allows for quantification of the changes in cytoskeletal dynamics caused by mutations in actin. Using this technique, quantification of cytoskeletal function in live cells valuably complements in vitro studies of protein function. As an example, missense mutations affecting the actin residue R256 have been identified in three human actin isoforms suggesting this amino acid plays an important role in regulatory interactions. The effects of the actin mutation R256H on cytoskeletal movements were studied using the yeast model. The protein, Aip1, which is known to assist cofilin in actin depolymerization, was tagged with green fluorescent protein (GFP) at the N-terminus and tracked in vivo using TIRF microscopy. The rate of Aip1p movement in both wild type and mutant strains was quantified. In cells expressing R256H mutant actin, Aip1p motion is restricted and the rate of movement is nearly half the speed measured in wild type cells (0.88 &#177; 0.30 &#956;m/sec in R256H cells compared to 1.60 &#177; 0.42 &#956;m/sec in wild type cells, p &#60; 0.005).

Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H.

Aip1p Dinámica se alteran por la R256H Mutación de actina

Mutations in actin cause a range of human diseases due to specific molecular changes that often alter cytoskeletal function. In this study, imaging of ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Triage de tejidos y de congelación para los modelos de la enfermedad del músculo esquelético

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Tandem congelación de alta presión y la rápida congelación de sustitución de tejidos vegetales para Microscopía Electrónica de Transmisión

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

La congelación de inmersión: una herramienta para los estudios ultraestructurales e inmunolocalización de células en suspensión en Microscopía Electrónica de Transmisión

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

La ingestión de fluorescente, nanopartículas magnéticas para determinar capacidades de absorción de fluidos en insectos

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...