CN113702148A

CN113702148A - Freezing embedding agent and application thereof in frozen section

Info

Publication number: CN113702148A
Application number: CN202110997603.3A
Authority: CN
Inventors: 陈莉莉; 尹盈; 唐清明; 陈广进; 彭金枫; 孙纪威; 罗贝贝
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2021-08-27
Filing date: 2021-08-27
Publication date: 2021-11-26

Abstract

The invention relates to the technical field of tissue sections, in particular to a frozen embedding agent and its application in frozen sections. The freezing embedding agent, in parts by weight, includes 10-30 parts of sucrose, 1-5 parts of polyvinylpyrrolidone and 3-10 parts of gelatin. In the invention, gelatin, sucrose and polyvinylpyrrolidone are used in the freezing embedding medium, and the characteristics of the three substances are fully exerted. It has a hardness similar to that of bone tissue, and the cell structure is not damaged during the quick freezing process. The bone tissue embedded in the freezing embedding agent provided by the present invention has intact tissue after being frozen sectioned, and exhibits a more complete structure during subsequent tissue staining.

Description

Freezing embedding agent and application thereof in frozen section

Technical Field

The invention relates to the technical field of tissue slices, in particular to a frozen embedding medium and application thereof in frozen slices.

Background

Tissue section technique is a commonly used method for observing morphological structure of cell tissue, and is commonly used in pathology or forensic science to study, observe and judge morphological changes of cell tissue. At present, for bone tissues, the slicing method is mainly divided into three methods, one method is to carry out slicing after the bone tissues subjected to decalcification are embedded by paraffin; one is to carry out frozen section after the bone tissue after decalcification is embedded by using a frozen embedding medium; yet another approach is to perform hard tissue sectioning of bone tissue without decalcification.

The principle of the method lies in that paraffin is fully immersed into tissues through the steps of dehydration, transparence, paraffin embedding and the like, so that tissue blocks are hardened, the tissue blocks are favorably sliced, the original structure of the tissues can be perfectly preserved, and the method can be used for observing tissue images under a microscope.

The frozen section is a method for rapidly cooling tissues to a certain hardness under a low temperature condition and then slicing, the manufacturing process is quicker and simpler compared with a paraffin section, and the frozen section can be applied to rapid pathological diagnosis in an operation.

Hard tissue sections are a method of generating very thin complete sections from hard tissues or large specimens and then observing the sections, but the cost is high, the microstructure is not as fine as that of paraffin sections, the thickness of the sections is generally 10 μm, and the sections cannot present some staining forms such as bone marrow microvessels.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a freezing embedding medium and application thereof in frozen sections.

According to a first aspect, the invention provides a freezing embedding medium, which comprises 10-30 parts by weight of sucrose, 1-5 parts by weight of polyvinylpyrrolidone and 3-10 parts by weight of gelatin.

The coating further comprises 15-25 parts by weight of cane sugar, 2-5 parts by weight of polyvinylpyrrolidone and 5-10 parts by weight of gelatin.

Further, the method comprises the following steps of:

15-20% of cane sugar, 2-3% of polyvinylpyrrolidone, 5-8% of gelatin and the balance of water.

Wherein, the gelatin forms gel at room temperature, and forms a three-dimensional network structure when cooled to below 0 ℃, thereby effectively reducing the wrinkle phenomenon during the frozen section. However, the difference between the hardness of the formed gel and that of the bone tissue is large in a cooling state, and slicing is not facilitated. The invention further solves the problem through polyvinylpyrrolidone, and the polyvinylpyrrolidone has good dispersibility and film forming property and has thickening effect, so that the hardness of the embedding agent after quick freezing is close to the hardness of the decalcified bone tissue. In addition, the invention also adds sucrose which can inhibit the dynamic performance of water molecules, thereby inhibiting the growth of ice crystals and playing a good role of a freezing protective agent in the quick-freezing process.

In a second aspect, the invention provides a method of frozen sectioning, using said frozen embedding medium for embedding.

Further, the embedding comprises:

heating and thawing the frozen embedding medium; completely placing the sliced tissues in the freezing embedding medium and cooling to be gelatinous to obtain sliced tissue gel; and placing the section tissue gel in liquid nitrogen for 10-15 seconds.

Further, after the sliced tissue gel is placed on the surface of liquid nitrogen for 45-60 seconds, the sliced tissue gel is placed at the temperature of-20 ℃ to-25 ℃ for slicing.

The bone tissue after decalcification is placed in a freezing embedding medium (for example, the freezing section embedding medium is added into a freezing section embedding box) to be immersed in the bone tissue, and after the bone tissue forms gel at room temperature, the gel is attached to the surface of liquid nitrogen to be quickly frozen for 45-60 seconds until the embedding block is completely whitened, so that the bone tissue freezing time is effectively shortened, and the formation of ice crystals is reduced. The temperature range of 0 ℃ to-5 ℃ is the maximum ice crystal generation zone, the quick freezing time is too long, and when the embedded tissue is taken out from liquid nitrogen, the embedding agent is cracked due to the rapid temperature rise.

Further, heating to 55-65 ℃; and/or, the cooling is to 20-30 ℃.

Further, before the embedding, pretreatment is also included;

the pretreatment comprises the following steps: carrying out gradient dehydration treatment on the slice tissues for 6-12 hours by using a dehydrating agent 1, and carrying out gradient dehydration treatment on the slice tissues for 6-12 hours by using a dehydrating agent 2;

the dehydrating agent 1 comprises 15-20 parts by weight of cane sugar, and the dehydrating agent 2 comprises 25-30 parts by weight of cane sugar, 1-3 parts by weight of polyvinylpyrrolidone and 5-10 parts by weight of DMSO.

Wherein, the sucrose can reduce the water content in the bone tissue so as to reduce the formation of ice crystals in the quick-freezing process, the DMSO can further reduce the formation of ice crystals in cells in the quick-freezing process of the bone tissue, and the polyvinylpyrrolidone can penetrate into the bone tissue so as to improve the overall structural strength of the marrow cavity and reduce the possibility of the fragmentation of tissues in the marrow cavity in the process of frozen sectioning.

Further, the sliced tissue is decalcified bone tissue.

The invention further provides the application of the freezing embedding medium in the embedding of the frozen section of the bone tissue.

The invention has the following beneficial effects:

the invention uses gelatin, sucrose and polyvinylpyrrolidone in the frozen embedding medium, gives full play to the characteristics of the three substances, can obviously reduce the wrinkle phenomenon in slicing when being applied to frozen slices, simultaneously can have the hardness similar to that of bone tissues after being frozen, and does not damage the cell structure in the process of quick freezing.

The bone tissue embedded by the freezing embedding medium provided by the invention is not easy to wrinkle and break in the process of preparing a freezing section, the tissue is relatively complete and not easy to crack, the section with the thickness of 8-100 mu m can be cut, and the subsequent tissue staining presents a more complete structure, such as a bone tissue microvascular structure and a vascular reticular structure.

In addition, the tissue section prepared by the frozen section method provided by the invention has stronger adhesive capacity to a glass slide in the preparation process, also shows excellent anti-slide-off capacity on a pathological-grade frosted glass slide, and effectively reduces the occurrence of the anti-slide-off phenomenon in the subsequent tissue immunostaining.

The frozen section method provided by the invention has the advantages of low cost, non-toxic and harmless components of the frozen embedding medium, good biological safety, good immunogenicity retention of various proteins of bone tissues, contribution to subsequent immune tissue staining and capability of omitting the step of antigen repair.

Drawings

FIG. 1 is a schematic diagram of immunofluorescence observation results under a confocal microscope of a mouse femur frozen section provided in example 1 of the present invention; the microscope magnification was 100 ×.

FIG. 2 is a schematic diagram of immunofluorescence observation results under a confocal microscope of a mouse femur frozen section provided in example 1 of the present invention; the microscope magnification was 600 ×.

FIG. 3 is a schematic diagram of natural light observation results of a mouse femur frozen section under a confocal microscope according to example 1 of the present invention; the microscope magnification was 40 ×.

FIG. 4 is a schematic diagram of immunofluorescence observation results under a confocal microscope of a mouse femur frozen section provided in example 2 of the present invention; microscope magnification was 200 x.

FIG. 5 is a schematic diagram of the observation result of the frozen mouse femur section under confocal microscope according to example 2 of the present invention; the microscope magnification was 40 ×.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The reagents and instruments used in the following examples are commercially available unless otherwise specified.

Example 1

The embodiment provides a method for frozen sectioning, which specifically comprises the following steps:

1. taking bone tissues: the mice are sacrificed, bone tissues at the position of the thighbone are taken, attached soft tissues are stripped, and the mice are placed in 4% paraformaldehyde for fixation for 24 hours;

2. decalcification treatment: washing bone tissue with PBS for 3 times, transferring the bone tissue into 10% EDTA decalcification solution with volume 20 times, placing in a rotary shaking table, and replacing the decalcification solution every day for one week;

3. embedding pretreatment: embedding pretreatment: placing the tissue sample into a dehydrating agent 1 for soaking for 8 hours, and then placing the tissue sample into a dehydrating agent 2 for soaking for 8 hours, so that the damage of ice crystals generated in the quick-freezing process of the tissue sample to the tissue is reduced;

the dehydrating agent 1 comprises the following components in percentage by mass: 20 percent of cane sugar and the balance of deionized water, wherein the dehydrating agent 2 comprises the following components: 30% of sucrose, 5% of DMSO, 2% of polyvinylpyrrolidone and the balance of deionized water.

4. Quick-freezing embedding: placing the pretreated tissue sample into a frozen section embedding medium, sticking an embedding block on the surface of liquid nitrogen, quickly freezing for 45 seconds, and transferring to-20 ℃ for storage;

the frozen section embedding medium comprises the following components in percentage by mass: 15% of sucrose, 2% of polyvinylpyrrolidone, 5% of gelatin and the balance of deionized water.

5. Freezing and slicing: and (4) carrying out frozen sectioning on the embedded sample, and setting the temperature to be-20 ℃ to obtain a frozen tissue section.

6. Tissue staining: the obtained mouse femur frozen tissue sections are subjected to immunofluorescence staining with the staining indexes of CD31, EMCN and DAPI nuclear staining, and finally, the sections are sealed by using an anti-fluorescence quencher.

7. And (3) observation by a confocal microscope: the stained bone tissue sections were observed by confocal microscopy.

The slicing results are shown in fig. 1 and fig. 2, and the lumen-like structure of the blood vessel can be clearly seen in fig. 1 (low power microscope) and fig. 2 (high power microscope), which illustrates that the bone tissue embedded by the freezing embedding agent provided by the invention can present a relatively complete structure after the freezing slicing and the subsequent staining of the slices, thereby facilitating the subsequent pathological study.

In addition, the section obtained in the slicing process is shown in fig. 3, and as can be seen from fig. 3, the section obtained by the invention is complete in tissue and is not easy to break. In addition, in the present embodiment, in the slicing process, 20 slices are obtained, wherein 90% of the slices are complete, and under the same condition, the slice integrity rate (based on the fact that the slice can be used for staining and subsequent observation) of the commercially available OCT embedding agent (purchased from SAKURA) is 30-40%, which indicates that the slice integrity rate obtained by freezing the slices is significantly improved by the freezing embedding agent provided by the present invention.

Example 2

1. Taking bone tissues: the mice are sacrificed, bone tissues at the thighbone are taken, attached soft tissues are stripped, and the mice are placed in 4% paraformaldehyde for fixation for 24 hours;

3. embedding pretreatment: placing the tissue sample into a dehydrating agent 1 for soaking for 8 hours, and then placing the tissue sample into a dehydrating agent 2 for soaking for 8 hours, so that the damage of ice crystals generated in the quick-freezing process of the tissue sample to the tissue is reduced;

the frozen section embedding medium comprises the following components in percentage by mass: 20% of sucrose, 2% of polyvinylpyrrolidone, 8% of gelatin and the balance of deionized water.

7. And (4) microscopic observation: the stained bone tissue sections were observed by a microscope.

The staining result of the section is shown in fig. 4, and the vascular structure can be clearly seen from fig. 4, which shows that the bone tissue embedded by the freezing embedding medium provided by the invention can present a more complete structure after the freezing section and the subsequent section staining, thereby facilitating the subsequent pathological research.

In addition, the section obtained in the slicing process is shown in fig. 5, and as can be seen from fig. 5, the section obtained by the invention is complete in tissue and is not easy to break. In the slicing process, 20 slices are obtained in total, wherein 85% of the slices are complete.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A frozen embedding agent, characterized in that, in parts by weight, comprising 10-30 parts of sucrose, 1-5 parts of polyvinylpyrrolidone and 3-10 parts of gelatin.

2 . The frozen embedding agent according to claim 1 , wherein, in parts by weight, it comprises 15-25 parts of sucrose, 2-5 parts of polyvinylpyrrolidone and 5-10 parts of gelatin. 3 .

3. freezing embedding agent according to claim 1 and 2 is characterized in that, in mass percent, comprising:

15-20% of sucrose, 2-3% of polyvinylpyrrolidone and 5-8% of gelatin, and the balance is water.

4. A method for frozen section, characterized in that, embedding is carried out using the frozen embedding agent described in any one of claims 1-3.

5. The frozen section method according to claim 4, wherein the embedding comprises:

Heat and thaw the frozen embedding medium; place the sliced tissue completely in the frozen embedding medium and cool it to a gel-like state to obtain a sliced tissue gel; place the sliced tissue gel in liquid nitrogen for 10 to 15 seconds .

6. The method according to claim 4 or 5, wherein after placing the sliced tissue gel on the surface of liquid nitrogen for 45-60 seconds, the sliced tissue gel is placed at -20°C～- Sectioned at 25°C.

7. The frozen section method according to any one of claims 4-6, wherein the heating is heating to 55-65°C; and/or the cooling is cooling to 20-30°C.

8. The frozen section method according to any one of claims 4-7, characterized in that, before the embedding, further comprising pretreatment;

The pretreatment includes: placing the sliced tissue in a cryoprotectant for 6-12 hours;

The cryoprotectant includes, in parts by weight, 10-20 parts of DMSO and 1-3 parts of polyvinylpyrrolidone.

9. The frozen section method according to any one of claims 4-8, wherein the sectioned tissue is demineralized bone tissue.

10. The application of the cryo-embedding agent according to any one of claims 1-3 in the embedding of frozen sections of bone tissue.