This project was supported by the National Natural Science Foundation of China (NSFC):&#160;31301950 and 31671288.

The current method has been established and validated to harvest and store more than 1,000 muscle samples for histological staining in our laboratory. All procedures involving animal care and use followed the guidelines established by the Ministry of Agriculture of China.
1. Equipment for Sample Collection
<ol>
	<li>Label the sample identity on each cryogenic vial. 
	Note: The cryogenic vial is specially designed to freeze fresh tissue samples for the cryosection procedure (Figure 1A). The vials are made of polypropylene in a mold factory (Figure 1C).</li>
	<li>Obtain the following equipment: liquid nitrogen in an appropriate liquid nitrogen tank, safety glasses or a face shield, freezer gloves, 10 cm forceps, 25 cm tweezers, a Styrofoam cooler (inner dimensions: 20 cm long x 12 cm wide x 13.5 cm high; outer dimensions: 24 cm long x 16 cm wide x 15 cm high), plastic film (2.5 cm long x 0.8 cm wide x 0.1 mm thick), a scalpel, and A4 PVC binding covers (21 cm x 29.5 cm) (Figure 1B).</li>
</ol>
2. Preparation of Muscle Samples
<ol>
	<li>Fill the Styrofoam cooler with liquid nitrogen 5 min ahead of sample collection. 
	NOTE: The large column of liquid nitrogen is essential because part of the liquid nitrogen boils away and turns into gas when fresh samples touch it. In order to reduce the frequency of transfer of liquid nitrogen to replenish the loss, an appropriately sized Styrofoam cooler must be acquired in advance according to the number of samples.</li>
	<li>Once a specimen is obtained, gently place it on a piece of A4 PVC binding cover and observe the direction of the muscle fibers. 
	NOTE: Here, we used the cross-section of the longissimus doris muscle from the first to the last lumbar vertebra in pigs. The specimens are about 12 cm long, 8 cm wide, and 5 cm high.</li>
	<li>Use a scalpel to make two parallel incisions through the specimen in the direction of the muscle fibers, approximately 3 cm in length and 0.6 cm apart. Trim the incised muscle into a rectangle approximately 0.6 cm wide x 0.6 cm high x 1.5 cm long. 
	NOTE: The reliability of measuring the cross-sectional area of the muscle fibers mostly depends on the fiber orientation of the incised muscle.</li>
	<li>Use 10 cm forceps to gently put the sample on a piece of plastic film, with the longitudinal axis of the incised muscle parallel to the long edge of the film (Figure 2A). 
	NOTE: The plastic film has two functions: a holding function that helps in fixing the orientation of the muscle fibers and an adhesion function that prevents cracks in the sample due to rapid freezing.</li>
	<li>Using forceps, place the muscle tissue into the lower middle of a labelled cryogenic vial, just between the inlet holes, and then tightly cap the vial (Figure 2B).</li>
</ol>
3. Freezing and Storing Muscle Samples
<ol>
	<li>Using 25 cm tweezers, quickly submerge the whole cryogenic vial in liquid nitrogen in the pre-cooled Styrofoam cooler, waiting until the liquid nitrogen does not boil (approximately 10-20 s). 
	NOTE: The cryovial will float when the liquid nitrogen is boiling, so carefully immerse the vial in the boiling liquid nitrogen for 10-20 s.</li>
	<li>Transfer the muscle samples to a liquid nitrogen storage tank or a -80 &#176;C freezer for long-term storage.</li>
</ol>
4. Slide Preparation
<ol>
	<li>Precool a cryostat to between -20 &#176;C and -22 &#176;C.</li>
	<li>Remove and discard the old microtome blade, wipe down the knife holder and anti-roll plate in the cryostat, install a new microtome blade in the cryostat, and set the cutting thickness to 8 &#181;m.</li>
	<li>Transfer the muscle sample in the cryogenic vial from the liquid nitrogen storage tank or -80 &#176;C freezer to the cryostat, transporting it in liquid nitrogen or on dry ice.</li>
	<li>Wait 30 min for the specimen to equilibrate with the temperature of the cryostat. 
	NOTE: Any equipment that might touch the specimen must be pre-cooled, because a high temperature might cause the formation of ice crystals in the specimen.</li>
	<li>Mount the sample in the specimen holder under the optimum cutting temperature formulation of water-soluble glycols and resins (OCT). Cut 8-&#181;m sections. 
	NOTE: Adjust the cutting angle perpendicularly to the orientation of the muscle fibers, because the CSAF is the cross-sectional area of muscle fibers.</li>
	<li>Mount the sections towards the center of a room-temperature microslide. Immediately place the slide into a slide box in a -80 &#176;C freezer. Do not allow the slide to dry at RT.</li>
</ol>

<ol><li>Joo, S. T., Kim, G. D., Hwang, Y. H., Ryu, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+fresh+meat+quality+through+manipulation+of+muscle+fiber+characteristics%5BTitle%5D)+AND+%22Meat+Sci%22%5BJournal%5D)">Control of fresh meat quality through manipulation of muscle fiber characteristics.</a> Meat Sci. 95 (4), 828-836 (2013).</li>
<li>Lefaucheur, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+second+look+into+fibre+typing--relation+to+meat+quality%5BTitle%5D)+AND+%22Meat+Sci%22%5BJournal%5D)">A second look into fibre typing--relation to meat quality.</a> Meat Sci. 84 (2), 257-270 (2010).</li>
<li>Fiems, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Double+Muscling+in+Cattle%3A+Genes%2C+Husbandry%2C+Carcasses+and+Meat%5BTitle%5D)+AND+%22Animals+%28Basel%29%22%5BJournal%5D)">Double Muscling in Cattle: Genes, Husbandry, Carcasses and Meat.</a> Animals (Basel). 2 (3), 472-506 (2012).</li>
<li>Cockett, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+callipyge+mutation+and+other+genes+that+affect+muscle+hypertrophy+in+sheep%5BTitle%5D)+AND+%22Genet+Sel+Evol%22%5BJournal%5D)">The callipyge mutation and other genes that affect muscle hypertrophy in sheep.</a> Genet Sel Evol. 37, Suppl 1 65-81 (2005).</li>
<li>Stinckens, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RYR1+g.1843C%3ET+mutation+is+associated+with+the+effect+of+the+IGF2+intron3-g.3072G%3EA+mutation+on+muscle+hypertrophy%5BTitle%5D)+AND+%22Anim+Genet%22%5BJournal%5D)">The RYR1 g.1843C>T mutation is associated with the effect of the IGF2 intron3-g.3072G>A mutation on muscle hypertrophy.</a> Anim Genet. 38 (1), 67-71 (2007).</li>
<li>Brameld, J. M., Buttery, P. J., Dawson, J. M., Harper, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nutritional+and+hormonal+control+of+skeletal-muscle+cell+growth+and+differentiation%5BTitle%5D)+AND+%22Proc+Nutr+Soc%22%5BJournal%5D)">Nutritional and hormonal control of skeletal-muscle cell growth and differentiation.</a> Proc Nutr Soc. 57 (2), 207-217 (1998).</li>
<li>Reinoso, R. F., Telfer, B. A., Rowland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+water+content+in+rats+measured+by+desiccation%5BTitle%5D)+AND+%22J+Pharmacol+Toxicol+Methods%22%5BJournal%5D)">Tissue water content in rats measured by desiccation.</a> J Pharmacol Toxicol Methods. 38 (2), 87-92 (1997).</li>
<li>Meng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+triage+and+freezing+for+models+of+skeletal+muscle+disease%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Tissue triage and freezing for models of skeletal muscle disease.</a> J Vis Exp. (89), (2014).</li>
<li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do%27s+and+don%27ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J Vis Exp. (99), e52793(2015).</li>
<li>Meijer, A. E., Vloedman, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+histochemical+characterization+of+the+coupling+state+of+skeletal+muscle+mitochondria%5BTitle%5D)+AND+%22Histochemistry%22%5BJournal%5D)">The histochemical characterization of the coupling state of skeletal muscle mitochondria.</a> Histochemistry. 69 (3), 217-232 (1980).</li>
<li>Harnkarnsujarit, N., Kawai, K., Suzuki, T. <a target="_blank" href="http://agris.fao.org/agris-search/search.do?recordID=US201500156343">Effects of Freezing Temperature and Water Activity on Microstructure, Color, and Protein Conformation of Freeze-Dried Bluefin Tuna (Thunnus orientalis).</a> Food Bioprocess Technol. 8 (4), 916-925 (2015).</li>
<li>Dubowitz, V., Sewry, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aDubowitz%2C+V+%22Muscle+Biopsy%3A+A+Practical+Approach%22">Muscle Biopsy: A Practical Approach</a>. , Elsevier. Ch. 15 407-422 (2007).</li>
<li>Suvarna, S. K., Layton, C., Bancroft, J. D. <a target="_blank" href="http://www.elsevier.ca/ISBN/9780702042263/Bancrofts-Theory-and-Practice-of-Histological-Techniques">Bancroft's Theory and Practice of Histological Techniques: Expert Consult: Online and Print, 7e</a>. , Elsevier. (2008).</li>
<li>Sulaiman, S. A., Kamarudin, N. A. Z. <a target="_blank" href="https://www.researchgate.net/publication/222964817_Bubble_Size_in_Coalescence_Dominant_Regime_of_Turbulent_Air-Water_Flow_Through_Horizontal_Pipes">Bubbles Size Estimation in Liquid Flow Through a Vertical Pipe.</a> J Appl Sci. 12 (23), 2464-2468 (2012).</li>
<li>Fukutomi, K., et al. <a target="_blank" href="https://www.jstage.jst.go.jp/article/kikaib1979/53/496/53_496_3516/_article">A Study of A Flow through Small Apertures : 2nd Report, Experiments on The Velocity Field.</a> Nihon Kikai Gakkai Ronbunshu B Hen/transactions of the Japan Society of Mechanical Engineers Part B. 53 (496), 3516-3521 (1987).</li>
<li>Shah, M. S., Joshi, J. B., Kalsi, A. S., Prasad, C. S. R., Shukla, D. S. <a target="_blank" href="http://agris.fao.org/agris-search/search.do?recordID=US201500029141">Analysis of flow through an orifice meter: CFD simulation.</a> Chem Eng Sci. 71 (9), 300-309 (2012).</li>
</ol>

No conflicts of interest, financial or otherwise, are declared by the authors.

Here, we describe a new, multi-hole cryovial for freezing and storing muscle tissues to perform histological assessments of muscle function. The critical modified step in this protocol is that the sample in the multi-hole cryovial is directly immersed in liquid nitrogen. To our knowledge, this is the simplest and rapidest way to obtain excellent frozen samples for muscle cryosectioning among the existing freezing methods (see the representative results).
The technical challenge faced by muscle...

Skeletal muscle is the most valuable component of a meat-producing animal from the nutritional and processing point of view. In the meat industry, there are two especially critical aspects: the efficiency of muscle growth and the quality of the resulting meat. As a main component of the muscle, muscle fibers are directly related to growth performance and fresh meat quality in animals1. For example, the Total number of Fibers (TNF) and the Cross-sectional area of Fibers (CSAF) mostly determine muscle mass and meat quality; also, Fiber Type Composition (FTC) strongly affects fresh meat quality2. Therefore, the manipulation of muscle fiber characteristics in animals is a highly effective method to increase the core profitability and competitiveness of farms1.
To date, several intrinsic and extrinsic factors have been identified to manipulate muscle fiber characteristics1. This manipulation can be achieved through the targeted selection of animals with specific genes, such as the Myostatin gene in cattle3, the Callipyge gene in sheep4, and the RYR1 and IGF2 genes in pigs5. Also, diet control and treatments with specific hormones play an important role in muscle fiber characteristics6. Thus, an approach that combines genetic and nutritional factors might be able to improve lean meat content and meat quality. However, studies on muscle fibers are limited in the meat industry because the elucidation of the structure of muscle fibers still is a challenge.
Muscle fiber properties are identified using histochemical methods, such as the myosin adenosine triphosphatase (ATPase) assay. This method relies on the fact that enzymes located in thin (6-8 &#181;m) frozen sections of muscle fibers can be chemically reacted with certain products. However, the water content of muscles is greater than 75% in pigs, rabbits, mice, and humans, regardless of the position (i.e., back, abdomen, or hindlimb)7. Such high moisture content in muscles causes a commonly encountered issue &#8211; freezing artifacts &#8211; during the preparation of cryosections, as previously described8,9. In most cases, it is almost impossible to appropriately freeze muscle tissues in a slaughterhouse production line, according to our experience.
The protocol presented here describes a simple and efficient method used in our laboratory to freeze muscle tissues for cryosectioning in a high-throughput manner. The highlight of this method is a new cryovial that is designed for flash-freezing muscle tissues in liquid nitrogen. The current workflow can concurrently facilitate tissue freezing and processing for an excellent muscle cryosection, with a clearly visible cytoplasmic compartment and the tight apposition of myofibers to the surrounding tissue. In addition, this protocol can be applied to a wide array of options for tissue analysis because liquid nitrogen does not mix with tissues.

<table><tbody><tr><td>Cryostat Microtome</td><td>&amp;nbsp;Leica</td><td>Leica CM1950</td><td></td></tr><tr><td>Digital Microscope&amp;nbsp;</td><td>Nikon</td><td>Nikon DS-U3</td><td></td></tr><tr><td>&amp;nbsp;Cryogenic Vial&amp;nbsp;&amp;nbsp; Plastic film</td><td></td><td></td><td>Designed by ourself</td></tr><tr><td>Liquid Nitrogen</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>Scalpel</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>10 cm forceps</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>25 cm tweezer</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>Safety glass</td><td></td><td></td><td>Commomly-used</td></tr><tr><td>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

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

The cryovial illustration and the common laboratory equipment for freezing muscles during the preparation of frozen sections are shown in Figure 1. The cryovials are made of polypropylene in a mold factory. Each vial has a total of 14 inlet holes: one is on the cap; another is at the bottom; and the remaining 12 form four parallel lines, each with four holes at 90&#176; to one another. These inlet holes can speed up the flow velocity of liquid nitrogen next to the tissue ...

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

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

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

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Biology

11.3K Views.  Jiangxi Agricultural University. This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the "blanket effect" of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

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

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

Biochemistry

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures for rat laryngeal dissection, flash-freezing, and cryosectioning of the vocal folds. This study describes how to cryosection vocal folds in both longitudinal and cross-sectional planes. A novelty of this protocol is the laryngeal tracking during cryosectioning that ensures accurate identification of the intrinsic laryngeal muscles and reduces the chance of tissue loss. Figures demonstrate the progressive cryosectioning in both planes. Twenty-nine rat hemi-larynges were cryosectioned and tracked from the emergence of the thyroid cartilage to the appearance of the first section that included the full vocal fold. The full vocal fold was visualized for all animals in both planes. There was high variability in the distance from the appearance of the thyroid cartilage to the appearance of the full vocal fold in both planes. Weight was not correlated to depth of laryngeal landmarks, suggesting individual variability and other factors related to tissue preparation may be responsible for the high variability in the appearance of landmarks during sectioning. This study details a methodology and presents morphological data for preparing the rat vocal fold for histochemical neuromuscular investigation. Due to high individual variability, laryngeal landmarks should be closely tracked during cryosectioning to prevent oversectioning tissue and tissue loss. The use of a consistent methodology, including adequate tissue preparation and awareness of landmarks within the rat larynx, will assist with consistent results across studies and aid new researchers interested in using the rat vocal fold as a model to investigate laryngeal neuromuscular mechanisms.

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Neuroscience

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.

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.

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

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Medicine

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.

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

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Regulating Temperature in the Lab: Preserving Samples Using Cold

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs. This video will discuss some of the methods for keeping laboratory samples cold and will explain the correct cooling method to use for each experimental requirement.
For example, cooling agents, such as ice and dry ice, are typically used when keeping samples cold during experiments. This video discusses the physical properties of the most commonly used cooling agents, as well as safety precautions for working with them.
When it comes to keeping samples cold in between experiments, cooling equipment, including laboratory grade refrigerators and freezers can be used to preserve samples for extended period of time. Also discussed in this video are types of samples and reagents that can be stored in the commonly-available laboratory cooling equipment.
Finally, the concept of cryopreservation is introduced as a process through which tissues, cells, and biomolecules are cooled to sub-zero temperatures, thereby effectively stopping all sample-degrading biological activity. Several methods of cryopreservation are discussed that minimize or eliminate the formation of damaging ice crystals.

Preserving Samples Using Cold: Cooling Agents and Instruments, and Cryopreservation

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs.  This video will ...

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Snap Freezing of Muscle Tissue for Sectioning: A Protocol to Obtain Ultrathin Sections of Rapidly Frozen Murine Muscle Tissue

Source: Bonetto, A. et al. <a href="https://www.jove.com/video/54893" target="_blank">The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia.</a> J. Vis. Exp. (2016)
This video describes the procedure for snap freezing muscle tissue and obtaining its thin sections. The technique can help study cancer-related cachexia in colon cancer mouse models.