CN115226707B - Method for frozen section of low-temperature frozen tissue and application thereof - Google Patents

Abstract

The invention belongs to the technical field of frozen tissue slicing, and particularly relates to a method for frozen slicing of low-temperature frozen tissue and application thereof, wherein the method comprises the steps of programmed re-heating, embedding and slicing, including preparing a transfer box in advance, and pre-cooling for one night at-80 ℃ in a refrigerator for later use; taking out the tissue frozen at low temperature, placing the tissue into a transfer box, placing the transfer box into a refrigerator at-80 ℃ for 0.5-1h, and placing the transfer box into a refrigerator at-40 ℃ for 0.5-1h; the tissue is taken out and embedded on dry ice, and the slice is completed after being balanced for 0.5 to 1 hour in an ice cutting machine. According to the invention, the frozen non-embedded tissue is taken out from the storage place and subjected to programmed re-warming, and then frozen embedding and slicing are carried out, so that the problems of tissue fragmentation and breakage when the frozen non-embedded tissue is applied to frozen tissue slicing experiments can be effectively solved, and the frozen tissue slices subjected to re-warming treatment have good integrity, good shape and uniformity; the shape is good after dyeing, and the tissue structure is clear.

Description

A method and application of cryogenically cryopreserved tissue for frozen sectioning

Technical field

The invention belongs to the technical field of frozen tissue sectioning, and specifically relates to a method and application of cryogenically frozen tissue for frozen sectioning.

Background technique

Frozen section is a pathological experiment technology that uses low-temperature freezing to quickly freeze or embed living tissue to a certain hardness and then slice it. It is widely used in clinical and scientific research. Frozen section technology can quickly prepare section specimens and better preserve the enzyme activity in cells. At the same time, because frozen sections are less destructive to antigens and have no obvious shrinkage of tissues, they can preserve the activities of fats, lipids and various enzymes. They are also widely used in immunohistochemistry or immunofluorescence experiments and have an irreplaceable role in scientific research experiments. role. To produce high-quality frozen sections, it is necessary to fully master the technical essentials and control each operation step in every aspect of material collection, tissue freezing and embedding, slicing, and staining. The quality of frozen sections directly affects the experimental results. Therefore, making high-quality frozen sections is the key to obtaining high-quality experimental results. During the freezing process of tissues, due to the influence of water content, freezing time and temperature, the frozen sections may have problems such as ice crystal formation, section wrinkles, section detachment, and poor staining. The storage conditions of tissue specimens will also have an impact on the tissue. The antigens and tissue morphology of frozen sections will have a certain impact.

Currently, cryo-embedded tissues are generally used for sectioning in frozen section operations. The embedding agent can be OCT or glue. Generally, the frozen embedded tissue is sectioned immediately, or the embedded tissue sample is stored in a -80°C ultra-low temperature refrigerator. When slicing, the sample is taken out and transferred to an ice cutting machine to buffer for 30 minutes before slicing. However, sometimes because frozen sections were not considered before the experiment or due to time urgency or large amounts of materials, some tissues were not frozen and embedded in time, but were directly wrapped in cryovials or tin foil and placed in liquid nitrogen or -80 ℃ ultra-low temperature refrigerator. If such cryopreserved tissues are directly taken out for embedding and slicing, tissue slices will be easily torn and damaged, affecting tissue morphology, interfering with the accuracy of staining results, and easily causing misjudgments, which will have certain consequences for pathological diagnosis or scientific research. adverse effects.

Contents of the invention

In the existing technology, the tissue taken is put into liquid nitrogen or frozen in a -80°C refrigerator without being embedded. When used, it is taken out and directly frozen, embedded and sliced, which will cause problems such as fragmentation, damage and uneven staining of the tissue slices. . The invention provides a method and application for carrying out programmed rewarming of unembedded cryopreserved tissue before embedding slices. That is, after the unembedded tissue cryopreserved is taken out, a series of programmed procedures are performed before embedding. The step of rewarming followed by frozen sectioning can effectively solve the problems of tissue fragmentation and damage that occur when cryopreserved unembedded tissues are used in frozen tissue sectioning experiments.

In order to achieve the above purpose, the present invention provides a method for cryogenic sectioning of cryopreserved tissue, which includes programmed rewarming, embedding and sectioning, and specifically includes the following steps:

(1)Procedural rewarming:

a. Prepare the transfer box in advance and pre-cool it in a -80°C refrigerator for one night and set aside;

b. Take out the cryopreserved tissue and place it in the pre-processed transfer box;

c. Place the transfer box in a -80°C refrigerator for 0.5-1h;

d. Place the transfer box in a -40°C refrigerator for 0.5-1h;

(2) Embedding: Take out the tissue after programmed rewarming and embed it with embedding agent on dry ice;

(3) Slicing: Place the embedded tissue in an ice cutting machine to balance for 0.5-1 hour before slicing.

The principle of programmed rewarming is to gradually change the tissue storage environment so that the tissue temperature changes gradually and evenly without rising sharply due to changes in the environment. This process must strictly control the temperature of the cryopreserved non-embedded tissue during the rewarming process, so that the temperature rises evenly, and no sharp temperature rise (too large temperature span) or uneven heating will cause the formation of ice crystals and other problems to ensure that the tissue It can have better shape after slicing.

Under normal circumstances, unembedded cryopreserved tissues will be directly transferred from the cryogenic storage location to dry ice for embedding without a rewarming step. This method will cause fragmentation and damage of frozen sections and subsequent staining. Inequality problem. In order to solve the above problems and achieve the purpose of achieving good shape and uniform staining of cryopreserved unembedded cryopreserved tissue after sectioning of frozen tissue, the inventors conducted a large number of experiments and found that by removing the unembedded cryopreserved tissue from storage Afterwards, programmed rewarming is performed. After the frozen tissue is taken out, it is first placed in a pre-processed transfer box, and the transfer box is placed in a -80°C refrigerator for a period of time, and then placed in a -40°C refrigerator for a period of time. Then embedding and slicing are carried out. The frozen tissue undergoes a programmed rewarming process. Ice crystals will not be formed due to sudden changes in ambient temperature. This can effectively solve the above problems. The obtained frozen tissue sections are complete and in good shape. After staining , the tissue structure is clear, and it can basically achieve the effect of directly freezing and immediately slicing the embedded tissue.

Further, in the above technical solution, the cryopreserved tissue is an unembedded tissue preserved in liquid nitrogen or an unembedded tissue preserved at -80°C.

Furthermore, in the above technical solution, when the cryopreserved tissue is an unembedded tissue preserved in liquid nitrogen, the pretreated transport box is placed in liquid nitrogen for 1.5-2.5 hours before transporting the tissue. In this technical solution, placing the cell transfer box in liquid nitrogen for pretreatment before use can make the environment of the cell transfer box closer to liquid nitrogen, and the frozen tissue will not be affected by rapid changes in the environment after being taken out.

Further, in the above technical solution, the temperature change rate of the transfer box is ≤1°C/min, and the transfer box is a cell transfer box or a magnetic bead transfer box. The present invention selects a transfer box with a relatively slow temperature change rate, which can make the temperature change of the frozen tissue slow and uniform during the rewarming process, and will not affect the internal structural changes of the tissue due to sudden temperature changes.

Further, in the above technical solution, the embedding agent is OCT.

Further, in the above technical solution, the thickness of the slice is 6-10 μm.

The present invention also provides an application of the above-mentioned improved method in cryogenically cryopreserved non-embedded tissue for frozen tissue sectioning. This improved method can also be used for other cryopreserved non-embedded tissues, and is not limited to liquid nitrogen preservation or -80°C preservation of non-embedded tissues.

The beneficial effects of the present invention are:

The present invention can effectively solve the problem of using cryopreserved unembedded tissue in frozen tissue sectioning experiments by taking out the cryopreserved non-embedded tissue from the storage place, performing programmed rewarming, and then performing frozen embedding and slicing. To solve the problem of tissue fragmentation and damage, the frozen tissue sections after rewarming have good integrity, good shape and uniformity;

The improved method of the present invention is simple to operate, and the frozen tissue sections of unembedded tissue cryopreserved can be stained in good shape and the tissue structure can be clearly displayed;

The method of the present invention is used for some tissues that cannot be frozen and embedded in time due to reasons such as not considering frozen sectioning before the experiment, or due to time urgency, or excessive amount of material taken, and are directly wrapped in cryopreservation tubes or tin foil and placed in liquid. Materials stored in nitrogen or -80°C ultra-low temperature refrigerator can avoid tearing and damage of tissue sections, solve the problem of material distortion for pathological diagnosis or scientific research, and improve the accuracy of judgment.

Description of drawings

Figure 1 is a photomicrograph of stained frozen sections under a 100x microscope in Example 1 of the present invention;

Figure 2 is a photomicrograph of stained frozen sections under a 100x microscope in Example 2 of the present invention;

Figure 3 is a photomicrograph of stained frozen sections in Comparative Example 1 under a 100x microscope.

Detailed ways

The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the raw materials involved in the following examples are all common commercial products and can be purchased in the market.

The non-embedded tissues in the following examples are all mouse livers.

The present invention will be described in further detail below in conjunction with the examples:

Example 1

A method for frozen sectioning of unembedded tissue stored in liquid nitrogen, including the following specific steps:

(1)Procedural rewarming:

a. Prepare the cell transfer box in advance and pre-cool it in a -80°C refrigerator overnight for later use;

b. Pre-treat the cell transfer box in liquid nitrogen for 2 hours before use, take out the cryopreserved tissue and place it in the pre-treated cell transfer box;

c. Place the cell transfer box in a -80°C refrigerator for 0.5h;

d. Place the cell transfer box in a -40°C refrigerator for 0.5h;

(2) Embedding: Take out the tissue after programmed rewarming and embed it on dry ice with the embedding agent OCT;

(3) Slicing: Place the embedded tissue in an ice cutting machine to balance for 0.5 hours before slicing.

After the obtained sections were stained by conventional methods, they were observed and photographed under a 100x microscope. The photographs are shown in Figure 1. As can be seen from Figure 1, the stained sections have high integrity, good and uniform shape, and clear tissue structure.

Example 2

A method for frozen sectioning of unembedded tissue stored in a -80°C refrigerator, including the following specific steps:

(1)Procedural rewarming:

a. Prepare the magnetic bead transfer box in advance and pre-cool it in a -80°C refrigerator for one night and set aside;

b. Take out the cryopreserved tissue and place it in the pre-processed magnetic bead transfer box;

c. Place the magnetic bead transfer box in a -80°C refrigerator for 1 hour;

d. Place the magnetic bead transfer box in a -40°C refrigerator for 1 hour;

(2) Embedding: Take out the tissue after programmed rewarming and embed it on dry ice with the embedding agent OCT;

(3) Slicing: Place the embedded tissue in an ice cutting machine to balance for 0.5-1 hour before slicing.

After the obtained sections were stained by conventional methods, they were observed and photographed under a 100x microscope. The photographs are shown in Figure 2. As can be seen from Figure 2, the stained sections have high integrity, good and uniform shape, and clear tissue structure.

Comparative example 1

A method for frozen sectioning of unembedded tissue stored in a -80°C refrigerator, including the following specific steps:

(1) Embedding: Take out the unembedded tissue stored in the -80°C refrigerator and embed it on dry ice with the embedding agent OCT;

(2) Slicing: Place the embedded tissue in an ice cutting machine to balance for 1 hour before slicing.

After the obtained sections were stained by conventional methods, they were observed and photographed under a 100x microscope. The photographs are shown in Figure 3. As can be seen from Figure 3, the integrity of the stained sections is poor, with tears and damage, uneven shapes, and overlapping or missing tissue structures.

Based on the above results, it is shown that the present invention can effectively solve the problem of using cryopreserved unembedded tissues for freezing by taking out cryopreserved non-embedded tissues from storage, performing programmed rewarming, and then freezing, embedding and slicing. Problems of tissue fragmentation and damage occur during tissue sectioning experiments. Frozen tissue sections after rewarming have good integrity, good shape, and uniformity; after staining, the shape is good, and the tissue structure is clearly displayed. It can be sectioned immediately compared to directly frozen and embedded tissue. The effect is basically the same. This improved method can be applied to non-embedded tissue cryopreserved in frozen tissue sections. The effect is good and can be used as a rescue method for subsequent cryopreserved tissue morphology.

Finally, it should be emphasized that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications, as long as they are within the spirit of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the principles of the invention shall be included in the protection scope of the present invention.

Claims (6)

1. A method for cryosectioning of cryopreserved tissue, which is characterized by including programmed rewarming, embedding and sectioning, specifically including the following steps: (1)Procedural rewarming: a. Prepare the transfer box in advance and pre-cool it in a -80°C refrigerator overnight for later use; b. Take out the cryopreserved tissue and place it in the pre-processed transfer box; c. Place the transfer box in a -80°C refrigerator for 0.5-1h; d. Place the transfer box in a -40°C refrigerator for 0.5-1h; (2) Embedding: Take out the tissue after programmed rewarming and embed it with embedding agent on dry ice; (3) Slicing: Place the embedded tissue in an ice cutting machine to balance for 0.5-1 hour before slicing; The cryopreserved tissue is an unembedded tissue stored in liquid nitrogen or an unembedded tissue stored at -80°C; The temperature change rate of the transfer box is ≤1°C/min. 2. A method for cryosectioning of cryopreserved tissue according to claim 1, characterized in that when the cryopreserved tissue is an unembedded tissue preserved in liquid nitrogen, a pre-processed transfer box is used before transporting the tissue. Then put it into liquid nitrogen for 1.5-2.5h. 3. A method for cryosectioning of cryopreserved tissue according to claim 1, characterized in that the transport box is a cell transport box or a magnetic bead transport box. 4. The method for cryosectioning of cryopreserved tissue according to claim 1, wherein the embedding agent is OCT. 5. A method for performing frozen sectioning of cryopreserved tissue according to claim 1, characterized in that the thickness of the section is 6-10 μm. 6. Application of the method according to any one of claims 1 to 5 in cryogenically cryopreserved non-embedded tissue for use in frozen tissue sections.