Name: a multi-hole cryovial eliminates freezing artifacts when muscle tissues are directly immersed in liquid nitrogen
Uploaded: 2017-04-06T14:00:00
Description: 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

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
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	<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 ...

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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.

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			<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>
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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 ...

The overall goal of this procedure is to freeze muscle tissues, by plunging them directly into liquid nitrogen while eliminating freezing artifacts. Maozhang He, a student from my laboratory will demonstrate this procedure with Yizhong Huang. To begin, label the sample identity on each cryogenic vial.Then, organize the following equipment:a tank of liquid nitrogen, safety glasses or a face shield, freezer gloves, 10 centimeter forceps, and 25 centimeter tweezers. Also, organize a styrofoam cooler, plastic film, a scalpel, and A4 PVC binding covers. To prepare muscle samples, begin by filling the cooler with liquid nitrogen.Within five minutes of obtaining a specimen, gently place the sample on a piece of A4 PVC binding cover, and observe the direction of the muscle fibers. Next, use a scalpel to make two parallel incisions through the specimen in the direction of the muscle fibers, approximately 3 centimeters in length, and 0.6 centimeters apart. Trim the incised muscle into a rectangle approximately 0.6 centimeters wide, by 0.6 centimeters high, by 1.5 centimeters long.Then, using ten centimeter forceps, 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. Place the muscle tissue into the lower middle of a labeled cryogenic vial, just between the inlet holes, and tightly cap the vial. To freeze the tissue sample, use 25 centimeter tweezers to quickly submerge the whole cryogenic vial in liquid nitrogen, in the pre-cooled styrofoam cooler, waiting until the liquid nitrogen no longer boils.The liquid nitrogen should be adequate to immerse the whole cryovial, and the cryogenic vial should be entirely below the liquid level. Transfer the muscle samples to a liquid nitrogen storage tank, or a 80 degree celsius freezer for long-term storage. It is important to avoid accidental contact between the frozen muscles and the room temperature containers or instruments.To prepare slides, pre-cool a cryostat to between 20 degrees celsius, and 22 degrees celsius. Remove and discard the old microtone blade, and wipe down the knife holder, and anti-roll plate in the cryostat. Then, install a new microtone blade in the cryostat, and set the cutting thickness to eight micrometers.Remove the cryogenic vial with the muscle sample from the liquid nitrogen storage tank, or 80 degree celsius freezer, and place it in the liquid nitrogen, or on dry ice. Then, transfer the sample to the cryostat, and wait 30 minutes for the specimen to equilibrate with the temperature of the cryostat. After using OCT to embed the sample, mount the sample in the specimen holder.Then, cut eight micrometer sections. Mount the sections towards the center of a room temperature micro slide. Immediately place the slide into a slide box.Then, place the box of slides in a 80 degree celsius freezer. Do not allow the slide to dry at room temperature. As seen here, ice crystals formed when a sample of longissimus dorsi muscle from pigs was placed directly in a 80 degree celsius freezer, and caused a well known Swiss cheese effect on the muscle cryo-section.Ice crystals also formed when the muscle tissue was immersed directly in liquid nitrogen. Using another method, many needle-like ice crystals formed in the cytoplasmic compartment of myofibers when the tissue was dipped in OCT mounting medium in the proper orientation, and then rapidly frozen in a slurry of isopentane. Freezing the tissue into the multi-hole cryovial achieves excellent specimen integrity, with a clearly visible cytoplasmic compartment and the tight apposition of myofibers to the surrounding tissue.Using this method, ATPase activity was analyzed in fast-contracting, and slow-contracting muscle fibers. When given equivalent times, fast-contracting fibers appear light, with two degrees, and slow-contracting fibers appear black. In a standard freezing method shown here, muscle tissue was submerged in a slurry of isopentane without direct contact with OCT mounting medium, however a solid liquid slurry state of isopentane is difficult to maintain for more than one hour, and isopentane is not always available.

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