CN108918215B - Method for rapidly preparing hard tissue slices and application thereof - Google Patents
Method for rapidly preparing hard tissue slices and application thereof Download PDFInfo
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- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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Abstract
The invention relates to a method for rapidly preparing hard tissue slices, which comprises the following steps: fixing the hard tissue in paraformaldehyde, and then soaking in a sucrose aqueous solution; embedding the hard tissue by using an embedding agent, and then quickly freezing to solidify the embedding agent; closely adhering one side of the sample to the adhesive film strip, and then slicing the hard tissue along the direction parallel to the surface of the adhesive film strip at the temperature of minus 25 ℃ to minus 30 ℃, wherein the slicing thickness is 6-8 mu m; and (3) placing the membrane band with the hard tissue slices on an anti-falling glass slide, tightly attaching the hard tissue slices on the membrane band with the hard tissue slices to the anti-falling glass slide through ultraviolet curing glue, then initiating polymerization of the ultraviolet curing glue by using ultraviolet light, and removing the adhesive membrane band on the surface of the hard tissue slices to obtain the hard tissue slices. The invention also claims the application of the hard tissue section prepared by the method in tissue staining. The method is simple, convenient and easy to learn, low in price and quick (only 3 days).
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a method for quickly preparing a hard tissue slice and application thereof.
Background
Hard tissue (bone, tooth, etc.) diseases, especially orthopedic diseases such as osteoporosis, are common and serious harmful diseases. Tissue slice techniques are an important tool for studying pathological processes. The traditional method for slicing hard tissues such as bones generally comprises the steps of fixing, decalcifying, embedding and slicing the hard tissues, and then staining and observing the hard tissues. However, some important proteins are often denatured and some important signals are lost during the decalcification treatment process so that sensitive indexes are difficult to detect, thereby seriously affecting the experimental results. Furthermore, there are some important indicators (e.g. calcification status, bone formation dynamics parameters, etc.) that must be observed through hard tissue sections. The most commonly used hard tissue section method at present is the methyl methacrylate resin embedding method (MMA method). The method has the advantages of complex flow, high cost, long time consumption (2-3 weeks), special hard tissue microtomes and special tungsten steel blades (the price is more than 15000 yuan, the service life is limited), and high requirements on users. At present, only a few domestic units have expensive hard tissue microtomes, and the MMA method is difficult to popularize in China, so that the research on hard tissues in China is hindered to a certain extent.
Therefore, a method for preparing hard tissue slices, which is simple, easy to learn, short in time consumption, low in cost and independent of special equipment, is urgently needed.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for rapidly preparing a hard tissue section and application thereof, overcomes the defects of complex preparation process, high cost, long time consumption (2-3 weeks) and the like of the conventional hard tissue section method, and provides a method for preparing a hard tissue section, which is simple, convenient and easy to learn, low in price and rapid (only needs 3 days).
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides a method for rapidly preparing hard tissue sections, comprising the steps of:
(1) fixing the hard tissue in paraformaldehyde, and then soaking in a sucrose aqueous solution for 24-48 h;
(2) embedding the hard tissue processed in the step (1) by using an embedding medium, and then quickly freezing to solidify the embedding medium;
(3) tightly adhering one side of the sample treated in the step (2) to an adhesive film belt, and then slicing the hard tissue at-25 to-30 ℃ along the direction parallel to the surface of the adhesive film belt, wherein the slicing thickness is 6 to 8 mu m, so as to obtain the film belt with the hard tissue slices;
(4) and placing the membrane band with the hard tissue slices on an anti-falling glass slide, wherein the anti-falling glass slide is provided with ultraviolet curing glue, the hard tissue slices on the membrane band with the hard tissue slices are tightly attached to the anti-falling glass slide through the ultraviolet curing glue, and then, the ultraviolet curing glue is initiated to polymerize by ultraviolet light to remove the adhesive membrane band on the surface of the hard tissue slices, so that the hard tissue slices are obtained.
Further, in step (1), the hard tissue is a bone tissue.
Further, the hard tissue is a natural bone tissue or a tissue-engineered bone tissue. In a particular embodiment of the invention, the hard tissue is natural bone tissue of a mouse or rat.
Further, the hard tissue is a tibia or a femur.
Further, in the step (1), the concentration of paraformaldehyde is 4%.
Further, in step (1), the treatment temperature was 4 ℃.
Further, in step (1), after the hard tissue is fixed in paraformaldehyde, it is washed with PBS and then soaked with an aqueous sucrose solution.
Further, in the step (1), after the hard tissue is fixed in the paraformaldehyde, the hard tissue is firstly soaked in 10-20% (w/v) of sucrose aqueous solution for 6-24h, and then soaked in 30% (w/v) of sucrose aqueous solution for 12-24 h. The concentration of the sucrose aqueous solution is calculated by the mass ratio of sucrose to the volume of water.
Further, in the step (2), the embedding medium is OCT embedding medium, TBS frozen section embedding medium or Leica's cryogel (Cryo-gel) frozen section embedding medium.
Further, in the step (2), liquid nitrogen is used for quick freezing.
Further, in the step (3), the slice is performed using a freezing microtome. In a specific embodiment of the invention, the microtome is a Leica (Leica) microtome and the microtome blade is a 819 disposable narrow blade knife manufactured by Leica (Leica).
Further, in step (3), the roller is repeatedly rolled on the surface of the adhesive film tape several times so that one side of the sample processed in step (2) is closely adhered to the adhesive film tape.
Further, in the step (4), the detachment prevention adhesion slide glass is a positive charge detachment prevention adhesion slide glass.
Further, in the step (4), ultraviolet light emitted by an ultraviolet lamp is used for initiating the polymerization of the ultraviolet curing adhesive, and the time of ultraviolet irradiation is 30-60 seconds.
Further, in the step (4), the wavelength of the ultraviolet light is 312nm, and the power of the ultraviolet lamp is 48W.
In another aspect, the invention also claims a hard tissue slice prepared by the above method.
In yet another aspect, the invention also claims the application of the hard tissue section prepared by the method in tissue staining.
Further, hard tissue sections were used for Von Kossa staining, calcein fluorescence double labeling, immunohistochemical staining, alkaline phosphatase staining or tartrate-resistant acid phosphatase (TRAP) staining.
Further, Von Kossa staining was used to detect hard tissue calcification status, as follows:
(1) the hard tissue slices were left at room temperature for 30 minutes and then washed 3 times with deionized water for 5 minutes each;
(2) adding 5% silver nitrate, irradiating with strong light for 30 min, discarding silver nitrate solution, adding 5% sodium thiosulfate solution, and standing for 5 min;
(3) counter-dyeing with a nuclear fixed red dye solution, sealing and observing.
Further, the calcein fluorescence double-label is used for detecting dynamic parameters of bone formation, and the steps are as follows:
(1) injecting calcein (20mg/kg) into abdominal cavity of mouse 2 days and 7 days before collecting bone tissue, and preparing hard tissue slice according to the above method;
(2) the hard tissue sections were left at room temperature for 30 minutes and then washed 3 times with deionized water for 5 minutes each time;
(3) counter-staining with DAPI, sealing, and taking a picture with a fluorescence microscope for observation;
(4) and (4) calculating bone formation dynamic parameters.
The invention provides a rapid hard tissue slicing method by combining the membrane band transfer and ultraviolet curing technology on the basis of the conventional freezing slicing method, the method is simple, convenient and easy to learn, short in time consumption and low in cost, does not depend on special equipment, and the prepared hard tissue slice has a complete structure and high quality.
By the scheme, the invention at least has the following advantages:
(1) the time required for preparing hard tissue sections is short (only 3 days) compared to the conventional hard tissue section method of Methyl Methacrylate (MMA), which requires a long time (2-3 weeks).
(2) The method uses a common blade (<10 yuan/slice) and a conventional freezing and slicing machine, so that the required cost is low, and the operation is simple and easy to learn; conventional hard tissue sectioning techniques require special hard tissue microtomes and special tungsten steel blades (>1.5 ten thousand yuan/blade), which are expensive and difficult to operate (typically requiring a dedicated person to operate).
(3) The hard tissue section prepared by the method has complete structure and high quality, can keep the complete shape of bone marrow, does not need decalcification, and can be directly used for detecting the calcification condition (Von Kossa staining) and bone formation dynamic parameters (fluorescent double-label analysis).
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a schematic cross-sectional view showing a structure of a sample obtained by transferring a bone tissue slice from an adhesive film tape onto a glass slide by using an ultraviolet curing adhesive in example 1 of the present invention;
FIG. 2 shows calcein fluorescence double-labeling results of tibial hard tissue slices of mice in example 1 of the present invention;
FIG. 3 shows Von Kossa staining results of tibial hard tissue sections of mice in example 1 of the present invention;
FIG. 4 shows calcein fluorescence double-labeled results of rat femur hard tissue sections in example 2 of the present invention;
description of reference numerals:
1-adhesive film tape; 2-hard tissue; 3-ultraviolet curing glue; 4-anti-shedding glass slide.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the following examples of the present invention, the adhesive film tape used was an adhesive film tape (catalog No. 39475214) manufactured by Leica (Leica); the microtome is a Leica (Leica) microtome; the slicer knife is a 819 disposable narrow blade knife manufactured by Leica corporation; the ultraviolet curing glue is No. 63 ultraviolet curing glue produced by Nolan (Norland) company in the United states; the anti-dropping glass slide is a positive charge anti-dropping adhesion glass slide.
Example 13 preparation of tibial hard tissue sections of month-old mice
1. Mouse tibia tissue obtaining, fixing, embedding and liquid nitrogen quick freezing
(1) Killing the mouse, taking the tibia, removing redundant soft tissues, placing the tibia in precooled 4% Paraformaldehyde (PFA), and preserving and fixing the tibia at 4 ℃ overnight;
(2) taking out the fixed sample, washing the sample with PBS for 5 minutes for three times, then soaking the sample in 15% (w/v) sucrose solution for 1 hour, and then transferring the sample to 30% (w/v) sucrose solution for overnight at 4 ℃;
(3) placing the sample soaked by the sucrose in an embedding box containing an OCT embedding agent, adjusting the sample to a proper position, and making proper marks at the position needing to be sliced;
(4) placing a cell culture dish with the diameter of 10cm in a foam box containing liquid nitrogen, floating the culture dish on the liquid nitrogen, pre-cooling for 1 minute, and taking care to avoid the liquid nitrogen from entering the culture dish;
(5) placing the sample obtained in the step (3) in a precooled cell culture dish and quickly freezing until the OCT embedding medium is in a white solid state to obtain a quick-frozen bone tissue sample;
(6) the quick frozen bone tissue samples were sliced or temporarily stored in a-80 ℃ refrigerator as follows.
2. Frozen section
(1) Fixing the quick-frozen bone tissue sample on a freezing table of a freezing microtome by using OCT (optical coherence tomography), adjusting the temperature of a box body and the temperature of a machine head of the freezing microtome to-25 ℃, and reducing the temperature to-30 ℃ if the quick-frozen bone tissue sample is broken in the slicing process;
(2) fixing a freezing table containing a sample on a machine head of a freezing microtome and adjusting the direction to ensure that the sample is parallel to a blade as much as possible;
(3) slicing with a freezing microtome, discarding the unwanted part, cutting to a position close to the target area where hard tissue sections are to be prepared, taking out a piece of adhesive film tape, cutting the tape in half, and adhering one half of the adhesive film tape to the surface of the sample. Then, repeatedly rolling the surface of the adhesive film strip for several times from top to bottom by using a roller to remove air bubbles between the film strip and the sample, so that the film strip and the sample are more tightly adhered;
(4) the specimen was sliced slowly and at a constant speed in a direction parallel to the surface of the adhesive film tape to ensure that the cut tape had an intact bone tissue slice adhered thereto, the slice thickness being 8 μm.
3. Transferring the bone tissue slices from the adhesive film belt to a glass slide by using ultraviolet curing glue
(1) Dripping a drop of ultraviolet curing adhesive on the anti-drop glass slide to avoid bubbles as much as possible;
(2) placing the cut film tape slice adhered with the bone tissue slice on an anti-falling glass slide, wherein the sample face is downward and is directly contacted with the ultraviolet curing adhesive, so that the ultraviolet curing adhesive is slowly spread and filled between the whole sample and the anti-falling glass slide; referring to fig. 1, from top to bottom, an adhesive film tape 1, a hard tissue 2, an ultraviolet curing adhesive 3, and an anti-peeling glass slide 4 are sequentially arranged. Wherein the hard tissue 2 is mouse tibia.
(3) Placing the sample obtained in the step (2) on an ultraviolet lamp box for irradiation, thereby causing the ultraviolet curing adhesive to polymerize and crosslink, wherein the parameters of the ultraviolet lamp box are as follows: ultraviolet wavelength: 312 nm; ultraviolet power: 48W; power supply: 220V; the ultraviolet irradiation time is 60 seconds;
(4) the adherent membrane band was slowly removed with a sharp-tipped forceps, at which time the sample had been completely transferred from the adherent membrane band onto a glass slide, to give mouse tibial hard tissue sections, which were used directly in downstream experiments or temporarily stored in a-20 ℃ freezer.
The mouse tibia hard tissue section prepared above is subjected to calcein fluorescence color double labeling and Von Kossa staining, and the specific method is as follows:
A. calcein fluorescence double label:
(1) injecting calcein (20mg/kg) into abdominal cavity of mouse 2 days and 7 days before collecting bone tissue, and preparing hard tissue slice according to the above method;
(2) the hard tissue sections were left at room temperature for 30 minutes and then washed 3 times with deionized water for 5 minutes each time;
(3) counter-staining with DAPI, sealing, and taking a picture with a fluorescence microscope for observation; the results are shown in fig. 2, and fig. 2a-d represent fluorescence micrographs of different positions of the mouse tibia, wherein the cell nucleus marked by DAPI is blue, and the new bone marked by calcein fluorescence is green, and the results show that the mouse tibia slice obtained by the method has a complete bone and marrow structure, and the new bone marked by calcein fluorescence is clearly visible.
(4) The dynamic parameters of bone formation are calculated by bioquant software, and the results show that the mean value of the bone mineralization deposition rate (MAR) is 1.4 mu m/d, and the mean value of the bone formation rate (BFR/BS) is 1.25 mu m3/μm2The average of the/d and the bone mineralization surface ratio (MS/BS) was 88.9%.
B. Von Kossa staining:
(1) the hard tissue slices were left at room temperature for 30 minutes and then washed 3 times with deionized water for 5 minutes each;
(2) adding 5% silver nitrate, irradiating with strong light for 30 min, discarding silver nitrate solution, adding 5% sodium thiosulfate solution, and standing for 5 min;
(3) counter-dyeing with a nuclear fixed red dye solution, sealing and observing. The results are shown in FIG. 3, in which FIGS. 3a-d represent fluorescence micrographs of different positions of the tibia of the mouse, respectively, and in the images, the nucleus stained by the nuclear-fixing-red staining solution is red, and the calcified bone tissue is black, which indicates the mineralization of the tibia of the experimental mouse.
In the traditional slicing method, in the embedding operation process, the fixation, dehydration, transparence, embedding and solidification of the specimen all have obvious influence on the quality of the final slice. The procedure is complicated, the required time is long, and the time is 2 to 3 weeks. High cost, special blades and instruments, and special personnel operation.
Example preparation of hard tissue slices of femurs from aged 23-month rats
The mouse in example 1 was replaced with a rat, and the femur was collected, and other steps were performed according to example 1 to prepare rat femur hard tissue sections.
The results of calcein fluorescence color double labeling of rat femur hard tissue sections according to the method of example 1 are shown in fig. 4, where fig. 4a-d represent fluorescence micrographs of rat femur at different positions, respectively, in the figure, DAPI-labeled nuclei are blue, and calcein fluorescence-labeled new bone is green, and the results show that the rat femur sections obtained by the method have complete bone and marrow structures, and the calcein fluorescence-labeled new bone is clearly visible, which can completely satisfy downstream experimental analysis.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for rapidly preparing hard tissue slices is characterized by comprising the following steps:
(1) fixing the hard tissue in paraformaldehyde, firstly soaking in 10-20% (w/v) sucrose aqueous solution for 1h, and then soaking in 30% (w/v) sucrose aqueous solution for 12-24 h;
(2) embedding the hard tissue processed in the step (1) by using an embedding medium, and then quickly freezing to solidify the embedding medium;
(3) tightly pasting one side of the sample processed in the step (2) on the adhesive film tape, and then slicing the hard tissue at minus 25 ℃ to minus 30 ℃ along the direction parallel to the surface of the adhesive film tape, wherein the slicing thickness is 6-8 mu m, so as to obtain the film tape with the hard tissue slice;
(4) and placing the membrane band with the hard tissue slices on an anti-falling glass slide, wherein the anti-falling glass slide is provided with ultraviolet curing glue, the hard tissue slices on the membrane band with the hard tissue slices are tightly attached to the anti-falling glass slide through the ultraviolet curing glue, and then, the ultraviolet curing glue is initiated to polymerize by ultraviolet light to remove the adhesive membrane band on the surface of the hard tissue slices, so that the hard tissue slices are obtained.
2. The method of claim 1, wherein: in the step (1), the hard tissue is a bone tissue.
3. The method of claim 1, wherein: in step (2), the embedding medium is OCT embedding medium, TBS frozen section embedding medium or cryogel frozen section embedding medium.
4. The method of claim 1, wherein: in the step (2), liquid nitrogen is adopted for quick freezing.
5. The method of claim 1, wherein: in step (3), slicing is performed using a cryomicrotome.
6. The method of claim 1, wherein: in the step (4), ultraviolet light emitted by an ultraviolet lamp is used for initiating the polymerization of the ultraviolet curing adhesive, and the time of ultraviolet irradiation is 30-60 seconds.
7. A hard tissue slice prepared by the method of any one of claims 1-6.
8. Use of a hard tissue section prepared by the method of any one of claims 1-6 for tissue staining.
9. Use according to claim 8, characterized in that: the hard tissue sections were used for Von Kossa staining, calcein fluorescence double labeling, immunohistochemical staining, alkaline phosphatase staining or tartrate-resistant acid phosphatase staining.
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| CN112396924B (en) * | 2020-12-04 | 2022-02-18 | 南方医科大学 | Method for embedding skull |
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| CN114755407A (en) * | 2022-04-02 | 2022-07-15 | 北京大学深圳医院 | Preparation and detection method of human bone tissue multi-marker immunohistochemical sample |
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