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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. 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).
2. Obtain the following equipment: liquid nitrogen in an approp....

Results

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° to one another. These inlet holes can speed up the flow velocity of liquid nitrogen next to the tissue to av.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cryostat Microtome	Leica	Leica CM1950
Digital Microscope	Nikon	Nikon DS-U3
Cryogenic Vial   Plastic film			Designed by ourself
Liquid Nitrogen			Commomly-used
Scalpel			Commomly-used
10 cm forceps			Commomly-used
25 cm tweezer			Commomly-used
Safety glass			Commomly-used
Freezer gloves			Commomly-used