CN112414824B - Staining method for non-decalcified bone slice - Google Patents
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 16
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention belongs to the technical field of biomedicine, and particularly discloses a method for staining a non-decalcified bone slice, which comprises the following steps: slicing bone tissue with a diamond knife, grinding into bone slices with thickness of 15-25 μm with a grinding machine, dehydrating the bone slices, staining with Colophonium, and sealing with neutral gum to obtain stained non-decalcified bone slices. The tooth section prepared by the method has controllable manufacturing thickness and high quality, can well show the fine structure of the bone tissue, is more beneficial to identification, and can well meet the requirements of teaching and scientific research; the staining method of the invention is also suitable for the preparation of non-decalcified slices of other hard bone tissues.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a staining method of a non-decalcified bone slice.
Background
Morphology and histology are one of the important means for studying bone tissue behavior and bone repair and reconstruction processes. The standard method for detecting changes in bone tissue and obtaining experimental data is to perform embedded sectioning and staining analysis on bone tissue, with paraffin embedding being the most common method. Mature bone tissues (such as enamel, dentin, cementum and alveolar bone in teeth and periodontal tissues) are hard and dense in texture, difficult to cut, brittle and fragile, and need to be decalcified before paraffin embedding, and decalcification is a key step for determining the quality and staining effect of bone tissue slices such as teeth. However, the decalcification treatment seriously damages the inherent structure of bone tissue, and thus cannot distinguish between bone formation and mineralized bone and uncalcified osteoid, and cannot accurately evaluate the formation of new bone.
In order to be able to preserve the bone composition intact and to perform sectioning analyses, resin embedding methods without decalcification have been proposed. In the resin embedding method, the resin liquid is used for replacing paraffin, and the solidified resin specimen has tough texture and is not easy to crack, so that the resin specimen is suitable for section and analysis of non-decalcified bone tissues, such as plastic-embedded section and staining of non-decalcified bone tissues (LvRong et al, J. Clin & Experimental Pathology, p. 342). The non-decalcified bone tissue slice keeps the complete mineralized structure of the bone tissue, and the specimen can distinguish calcified bone from non-calcified bone after being stained. However, the conventional staining method, such as HE (hematoxylin-eosin) staining method, can destroy the microstructure of the bone tissue such as teeth, and cannot meet the teaching and scientific research requirements. And the bone slice without special staining is inconvenient for observing the morphological structure of the bone tissue.
In addition, at present, the non-decalcification bone slice is manufactured by a diamond cutter, but the thickness is generally 100 μm or more, and the slice thickness is thick, so that the study on bone tissue morphology is not facilitated; the traditional non-decalcification bone slice grinding method mainly comprises the steps of manually grinding, wherein the thickness of the plate is more than 40 mu m, the thickness of the edge and the center of the plate is uneven, the thickness of the plate is thicker, and the manual grinding is time-consuming and labor-consuming.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a method for staining a non-decalcified bone slice, which is used to solve the problem that the staining method of the non-decalcified bone slice in the prior art damages the microstructure of the bone tissue.
To achieve the above and other related objects, a first aspect of the present invention provides a method for staining a non-decalcified bone slice, comprising the steps of: slicing bone tissue with a diamond knife, grinding into bone slices with thickness of 15-25 μm with a grinding machine, dehydrating the bone slices, staining with Colophonium, and sealing with neutral gum to obtain stained non-decalcified bone slices.
Further, the dehydrated bone slices are fully soaked in the rosin baked into liquid state, and then immediately placed on a glass slide and sealed by neutral gum to obtain the dyed non-decalcified bone slices.
Further, the method comprises the steps of:
(1) cutting the bone tissue into bone slices with the thickness of 200-300 mu m by using a diamond cutter slicing machine;
(2) firmly sticking one side of the bone slice on an object carrying plate, finely polishing one side of the bone slice, which is not stuck on the object carrying plate, on a bone grinding machine, taking the bone slice off the object carrying plate, cleaning, firmly sticking the polished side of the bone slice on the object carrying plate, repeating the polishing process, polishing the non-polished side of the bone slice, wherein the thickness of the bone slice after being finely polished by the bone grinding machine is 15-25 mu m;
(3) taking the bone slices off the object carrying plate, cleaning, and dehydrating;
(4) baking rosin to liquid state, fully soaking the bone slices with the rosin, immediately placing the bone slices on a glass slide, and sealing the bone slices with neutral gum to obtain dyed non-decalcified bone slices.
Further, in the step (1), the rotating speed of the diamond knife slicing machine is 5000-.
Further, in the step (2), the bone slices are firmly adhered to the carrying plate by using the baked shellac.
Further, in the steps (2) and (3), the polished bone slices are placed in alkaline water, heated, taken down from the object carrying plate, removed of shellac, cleaned with clear water, and subjected to the next operation.
Further, the alkaline water is prepared from sodium carbonate, and the concentration of the sodium carbonate in the alkaline water is 5-10%.
Further, in the step (2), when the bone slice is ground on a bone grinding machine, the parallelism of the object carrying plate is 10 μm.
Further, in the step (2), the carrier plate is selected from one of a glass plate, a plastic plate and a stainless steel plate.
Further, in the step (3), the bone slices are dehydrated by using absolute ethyl alcohol.
Further, the bone tissue is compact bone.
Further, the compact bone is a tooth, and the bone slice is a tooth slice.
In a second aspect, the invention provides a non-decalcified bone slice prepared by the staining method of the first aspect.
Further, the non-decalcified bone slice is a non-decalcified tooth slice.
As described above, the staining method of the non-decalcified bone slice of the present invention has the following advantageous effects:
the invention provides a staining method of non-decalcification bone slices, which does not use embedding agents such as resin and the like in the process of preparing the bone slices, reduces the damage of the embedding agents with higher temperature to bone tissues and shows the microstructure of the bone tissues as intact as possible.
The invention firstly proposes to dye bone slices by rosin, and the principle of rosin dyeing is as follows: the rosin contains a certain amount of natural pigment, and after the bone slices are ground and stained by the rosin, various structural textures of bone tissues can have partial pigment deposition, so that the textures of enamel, dentin and cementum of the tooth body are observed to be enhanced, and the observation is more convenient compared with an undyed bone slice.
Experiments prove that the method for manufacturing the high-quality non-decalcification bone slice with controllable tooth slice thickness and visible microstructure can well meet the requirements of teaching and scientific research. The method of the invention is also suitable for non-decalcifying slicing and staining preparation of other bone tissues, particularly compact bones.
Drawings
FIG. 1 is a graph showing the results of rosin staining of non-decalcified dental sections in example 1 of the present invention.
Fig. 2 shows three connection patterns of the cementum enamel boundary in a textbook (oral histopathology, fourth edition of the national public health press, P23).
FIG. 3 is a tissue morphology diagram of an unstained section of non-decalcified teeth in comparative example 1 of the present invention.
FIG. 4 is a graph showing the results of HE staining of non-decalcified dental sections in comparative example 2 of the present invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The invention provides a staining method of non-decalcification bone slices, which comprises the steps of cutting bone tissues into slices with the thickness of 200-300 mu m by a diamond cutter slicing machine, polishing the slices into bone slices with the thickness of 15-25 mu m by a polishing machine, dehydrating the bone slices, staining the bone slices with rosin, and sealing the bone slices with neutral gum to obtain the stained non-decalcification bone slices.
The staining method of the non-decalcified bone slice specifically comprises the following steps:
(1) cutting the bone tissue into bone slices with the thickness of 200-300 mu m by using a diamond cutter slicing machine;
(2) firmly sticking one side of the bone slice on an object carrying plate, finely polishing one side of the bone slice, which is not stuck on the object carrying plate, on a bone grinding machine, taking the bone slice off the object carrying plate, cleaning, firmly sticking the polished side of the bone slice on the object carrying plate, repeating the polishing process, polishing the non-polished side of the bone slice, wherein the thickness of the bone slice after being finely polished by the bone grinding machine is 15-25 mu m;
(3) taking the bone slices off the object carrying plate, cleaning, and dehydrating;
(4) baking rosin to liquid state, fully soaking the bone slices with the rosin, immediately placing the bone slices on a glass slide, and sealing the bone slices with neutral gum to obtain dyed non-decalcified bone slices.
Specifically, in the step (1), the rotation speed of the diamond knife slicer is controlled at 5000-.
Specifically, in the step (2), the bone slices are firmly adhered to the carrying plate by using the baked shellac.
Specifically, in the steps (2) and (3), the polished bone slices are placed in alkaline water, heated, taken down from the object carrying plate, removed of shellac, cleaned with clear water, and subjected to the next operation. Wherein, the alkaline water is prepared by sodium hydroxide, the concentration of sodium carbonate in the alkaline water is preferably controlled to be 5-10%, and the sodium carbonate solution with 5% of alkaline water is adopted in the following embodiments.
Specifically, in the step (2), when the bone slices are ground on the bone slice grinding machine, the parallelism of the object carrying plate is controlled to be 10 microns, so that the thickness of the ground bone slices is uniform.
Specifically, in the step (2), the carrying plate is a glass plate, and can also be made of other materials, such as a stainless steel plate, a plastic plate, and the like.
Specifically, in the step (3), the bone slices are dehydrated by absolute ethyl alcohol (alcohol).
The invention firstly uses a diamond cutter slicing machine to slice the bone tissue into 200-300 mu m thick slices, and then uses a grinding machine to grind the bone tissue into 15-25 mu m thick slices, wherein the thickness is controlled at 15-25 mu m, which is more beneficial to the observation of the later bone tissue morphology. In the process of manufacturing the bone slice, embedding agents such as resin and the like are not used, so that the damage of the embedding agents with higher temperature to the bone tissue is reduced, and the microstructure of the bone tissue is shown as intact as possible.
The invention firstly proposes to dye bone slices by rosin, and the principle of rosin dyeing is as follows: the rosin contains a certain amount of natural pigment, and after the bone slices are ground and stained by the rosin, various structural textures of bone tissues can have partial pigment deposition, so that the textures of enamel, dentin and cementum of the tooth body are observed to be enhanced, and the observation is more convenient compared with an undyed bone slice.
The staining method of the present invention is suitable for preparing bone slices of compact bone tissue such as teeth, and the method of the present invention will be described in detail below using teeth as an example.
Example 1
A preparation and staining method of non-decalcified tooth slices comprises the following steps:
(1) the teeth were sliced into 200-300 μm thick sections using a diamond-blade slicer (XQPI-66A. phi.400 diamond slicer) at 6000 rpm.
(2) Adhering one side of each tooth slice to a glass plate by using baked shellac (purple natural resin), finely polishing the side of each tooth slice which is not adhered with the shellac on a grinding machine, boiling by using alkaline water (5% sodium carbonate), removing the shellac, and rinsing by using clear water; and sticking the polished side of the tooth section on a glass plate by using shellac, and repeating the polishing process on the non-polished side of the tooth section. During the polishing process, the parallelism of the glass plate was 10 μm. The thickness of the final polished dental section is controlled to be 15-25 μm.
(3) The shellac was removed with alkaline water (5% sodium carbonate), rinsed with clear water, and the dental sections were dehydrated with absolute ethanol.
(4) Baking rosin to liquid state, fully soaking the tooth slices in the rosin, immediately placing the tooth slices on a glass slide, and sealing the tooth slices with neutral gum to obtain dyed non-decalcified tooth slices.
FIG. 1 is a graph showing the results of rosin staining of non-decalcified dental sections.
As can be seen from fig. 1, the dental section prepared in example 1 perfectly shows the fine structure of the tooth, wherein the root cross section, toms granular layer, extrinsic cementum, internal dentin are observed in fig. 1-a; 1-B, pits, dentinal tubules and twisted glaze can be observed; FIG. 1-C shows the glazing panel, glazing shuttle, enamel dentinal junction; the enamel cementum boundary can be observed on the 1-D picture, and the 1-D picture is the same as the A picture in three connection form pictures (figures 1-28) of the enamel cementum boundary in a textbook (figure 2, oral histopathology, fourth edition of people's health press, P23), which shows that the dental structure presented by the model picture in the textbook can be observed on the dental section prepared by the method of the invention, which shows that the dental section prepared by the invention has high quality, the fine structure is obviously visible, and the requirements of teaching and scientific research can be well met.
Comparative example 1
Dental sections having a thickness in the range of 15 to 25 μm were prepared according to the method of example 1, and the polished dental sections were dehydrated and directly sealed with neutral gum.
FIG. 3 shows a histomorphology of unstained non-decalcified dental sections.
Comparative example 2
Dental sections with a thickness in the range of 15-25 μm were prepared according to the method of example 1, and the polished dental sections were dehydrated, stained with HE, and then mounted with neutral gum.
FIG. 4 shows a morphology of tissues of non-decalcified dental sections after HE staining.
As can be seen from the comparison of FIGS. 1, 3 and 4, the tissue morphology of the section of the undecalcified tooth shown in FIG. 1 has a better distinct contour of the tooth tissue and is better for identification, because the natural pigment contained in the rosin impregnates the section of the tooth, and the tissue contour of the less calcified part (higher organic matter content) of the hard tissue of the tooth has a higher affinity for the pigment than the highly calcified part, which makes the tissue contour more distinct and is better for identification.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (6)
1. A staining method of non-decalcified dental sections is characterized by comprising the following steps:
(1) cutting the teeth into 200-300 mu m thick teeth slices by a diamond cutter slicing machine;
(2) one side of each tooth slice is firmly adhered to the object carrying plate, one side of each tooth slice, which is not adhered to the object carrying plate, is finely ground on a grinding machine, each tooth slice is taken down from the object carrying plate, the tooth slices are cleaned, the ground side of each tooth slice is firmly adhered to the object carrying plate, the grinding process is repeated, the non-ground side of each tooth slice is ground, and the thickness of each tooth slice, which is finely ground by the grinding machine, is 15-25 mu m;
(3) taking down the dental sections from the object carrying plate, cleaning, and dehydrating with absolute ethyl alcohol;
(4) baking rosin to liquid state, then fully soaking the tooth section with the rosin, immediately placing on a glass slide, and sealing with neutral gum to obtain the dyed non-decalcified tooth section.
2. The staining method of non-decalcified dental sections according to claim 1, wherein: in the step (1), the rotating speed of the diamond knife slicing machine is 5000-7000 rpm;
and/or, in the step (2), the tooth slices are firmly stuck on the object carrying plate by using baked shellac.
3. The staining method of non-decalcified dental sections according to claim 2, wherein: and (3) putting the polished tooth slices into alkaline water, heating, taking down the tooth slices from the object carrying plate, removing the shellac, cleaning with clear water, and carrying out the next operation.
4. The staining method of non-decalcified dental sections according to claim 3, wherein: the alkaline water is prepared from sodium carbonate, and the concentration of the sodium carbonate in the alkaline water is 5-10%.
5. The staining method of non-decalcified dental sections according to claim 1, wherein: in the step (2), when the dental sections are polished on a polishing machine, the parallelism of the object carrying plate is 10 microns;
and/or, in the step (2), the carrying plate is selected from one of a glass plate, a plastic plate and a stainless steel plate.
6. A non-decalcified dental section produced by the staining method according to any one of claims 1 to 5.
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| CN113324822B (en) * | 2021-06-30 | 2022-06-24 | 艾佧科技(北京)有限公司 | HE staining method for bone tissue abrasive disc of implant material of resin embedding belt |
| CN115931517A (en) * | 2022-12-05 | 2023-04-07 | 中山大学附属第五医院 | Dyeing method of unwinding collagen in bone and cartilage |
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| CN101361974A (en) * | 2008-07-30 | 2009-02-11 | 广东医学院 | Medicine combination for preventing and treating osteoporosis |
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| CN106482997A (en) * | 2016-11-17 | 2017-03-08 | 兰州大学 | A kind of bone slice preparation method suitable for amphibious, reptiles species |
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