Bone tissue multiple special staining analysis method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a bone tissue multiple special staining analysis method and application thereof. The invention develops a multiple special staining method and a kit which can be suitable for bone tissues with different decalcification degrees and can display various tissue structure components on one slice, and related spectrum extraction and splitting analysis software is matched to finally realize morphological structure marking, segmentation and statistical analysis of the bone tissues.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a bone tissue multiple special staining analysis method and application thereof. Background Art

[0002] Bones and other calcium-containing hard tissues play an indispensable role in maintaining life activities, promoting physical health and development. From supporting the body to participating in metabolic regulation, to supporting immune function, the health of the skeletal system directly affects overall health. Therefore, studying and understanding bones and related diseases is of great significance to improving the quality of human life. However, the current research process of bone tissue faces the following difficulties:

[0003] 1. Challenges of decalcification: In experiments on morphological observation of bone tissue, due to the different densities of calcium salts and embedding agent paraffin, direct sectioning is generally not possible and decalcification is required. There are many types of decalcification solutions, and the fastest acting one is a simple acid decalcification solution, but it is very destructive to tissues, the time is difficult to control, and it is easy to over-decalcify. The structure and physical and chemical properties of samples after excessive decalcification with simple acids are severely damaged. In classic morphological staining experiments, the samples are lightly stained or not stained, and the various tissue cell components cannot be effectively distinguished.

[0004] 2. Limitations of staining technology: Although traditional staining techniques for bone tissue can distinguish between bone tissue and cartilage tissue, they are obviously insufficient in displaying other structural components. A single staining scheme is also difficult to meet the needs of modern pathology and biological research for the analysis of fine structure and functional diversity. In order to more comprehensively display the microstructure of the tissue, some composite staining methods attempt to achieve simultaneous display of multiple tissue components by cleverly combining different dyes. However, such methods usually require extremely complex operating steps. From the preliminary treatment of the sample to the sequential addition of dyes to the precise control of various staining conditions, each link requires a high degree of professional skills and meticulous operation. Moreover, the entire staining process often takes a long time, which also reduces the experimental efficiency to a certain extent. More importantly, the staining effect of such composite staining methods is often unstable. Due to the interaction of multiple dyes and complex operating procedures, even under the same experimental conditions, the staining results of different batches may vary greatly, making it difficult to repeatedly obtain consistent staining effects.

[0005] 3. Insufficient morphological analysis software: When conventional histopathology image analysis software is used for result analysis, it only relies on thresholds to distinguish positive signals, which can easily miss weak signals and has poor accuracy. The analysis method using spectral acquisition and signal extraction can split overlapping signals based on spectral curves, regardless of signal strength. At the same time, through software intelligent learning, it can achieve goals such as positive signal splitting, positive area statistics, and single / multi-channel quantification.

[0006] Therefore, there is an urgent need to develop a method that can effectively label, segment and statistically analyze the morphological structure of bone tissue. Summary of the invention

[0007] In order to overcome the defects existing in the prior art and solve the three problems of single existing staining results of bone tissue and calcium-containing hard tissue, light or no staining of over-acid decalcified samples and limitations of threshold segmentation technology of morphological analysis software, the purpose of the present invention is to develop a multiple special staining method that can be applied to bone tissues with different degrees of decalcification and can display multiple tissue structure components on one slice, and at the same time combine it with the existing spectral splitting software to provide a more accurate and comprehensive analysis method for bone tissue research.

[0008] First, in a first aspect, the present invention provides a bone tissue multiple special staining analysis method, the method comprising the following steps:

[0009] (1) Cartilage staining: staining with 5% Alcian blue solution (pH 2.5) for 20 min, followed by washing with 1% acetic acid;

[0010] (2) Nuclear staining: Weigert's iron hematoxylin staining for 10 min, washing with tap water, differentiation (adding differentiation solution to remove excess hematoxylin dye adsorbed by tissues other than the nucleus), and blueing;

[0011] (3) Bone staining: 5% naphthol green staining for 3 min, washed with 1% acetic acid;

[0012] (4) Collagen staining: staining with 0.05% Sirius red solution for 8 min, followed by washing with 1% acetic acid.

[0013] In one embodiment, the method further comprises: decalcification of bone tissue samples, paraffin embedding, paraffin sectioning, section dewaxing and hydration, acetic acid washing and other pre-staining steps.

[0014] In one embodiment, the decalcification method is to use 10% formic acid for decalcification.

[0015] In one embodiment, the method further comprises dehydrating the sections and sealing the sections with neutral gum after staining.

[0016] In one embodiment, the method further comprises using inform analysis software to perform spectral extraction on the staining results and perform data analysis according to the experimental purpose.

[0017] In a second aspect, the present invention provides a bone tissue multiple special staining analysis kit, the kit comprising 5% alcian blue solution, 1% acetic acid, Weigert's iron hematoxylin, 5% naphthol green solution, and 0.05% Sirius red solution.

[0018] In one embodiment, the kit is used according to the aforementioned analysis method.

[0019] In a third aspect, the present invention provides application of the aforementioned analysis method in analyzing the morphological structure of bone tissue.

[0020] In a fourth aspect, the present invention provides use of the aforementioned kit in analyzing the morphological structure of bone tissue.

[0021] In one embodiment, the kit is used in conjunction with inform analysis software.

[0022] Compared with the prior art, the present invention has the following beneficial technical effects:

[0023] 1. Wider applicability: The staining kits in the prior art usually have strict requirements on the decalcification degree of bone tissue and have a relatively narrow scope of application. However, the multiple special staining kit of the present invention can be applied to bone tissues with different degrees of decalcification, which greatly improves the versatility of the kit and can meet the analysis needs of more different types of bone tissue samples.

[0024] 2. Richer information: Traditional methods often have difficulty in displaying multiple tissue structural components on a single slice, requiring multiple staining and observations, which are cumbersome to operate and may introduce errors. The kit of the present invention can display multiple tissue structural components on a single slice, providing researchers with more comprehensive and intuitive bone tissue information, which helps to gain a deeper understanding of the complex structure and functional relationship of bone tissue.

[0025] 3. More accurate and efficient analysis: The existing technology may need to rely on a variety of complex instruments and methods when analyzing the morphological structure of bone tissue, and the analysis process is time-consuming and laborious. The present invention can achieve morphological structure labeling, segmentation and statistical analysis of bone tissue by matching relevant spectrum extraction and splitting analysis software, which not only improves the accuracy of analysis, but also greatly improves work efficiency, providing a more powerful tool for bone tissue research.

[0026] 4. Overall solution: The present invention provides a complete solution from staining kit to software analysis, organically combining staining and analysis, and providing a one-stop service for bone tissue research. Compared with the scattered methods and tools in the prior art, the overall solution of the present invention is more convenient and efficient, which helps to promote the development of bone tissue research. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0028] Figure 1 HE staining results;

[0029] Figure 2 This is the result of toluidine blue staining;

[0030] Figure 3 This is the result of Safranin O fast green staining;

[0031] Figure 4 This is the result of cell nucleus staining;

[0032] Figure 5 This is the result of cartilage staining;

[0033] Figure 6 This is the result of bone staining;

[0034] Figure 7 This is the result of collagen staining (bright field microscope);

[0035] Figure 8 This is the result of collagen staining (polarized light microscopy);

[0036] Fig. 9 Results of staining for nuclei and cartilage;

[0037] Fig.10 Results of staining for nuclei and hard bones;

[0038] Fig.11 Results of cell nucleus and collagen staining;

[0039] Fig.12 Staining results for cartilage and bone;

[0040] Fig.13 The staining results for cartilage and collagen;

[0041] Fig.14 The results of bone and collagen staining;

[0042] Fig.15 This is the result of multiple special staining of bone tissue (bright field microscope);

[0043] Fig.16 It is the result of multiple special staining of bone tissue (polarized light microscope);

[0044] Fig.17 It is the spectral information of the single dyeing results of 4 dye solutions;

[0045] Fig.18 Spectral splitting results for nuclei and cartilage staining;

[0046] Fig.19 Spectral splitting results for nuclei and bone staining;

[0047] Fig. 20The results of spectral splitting for nuclei and collagen staining;

[0048] Fig.21 Spectral splitting results for cartilage and bone staining;

[0049] Fig. 22 The results of spectral separation for cartilage and collagen staining;

[0050] Fig.23 The results of spectrum separation for bone and collagen staining;

[0051] Fig.24 This is the result of spectral decomposition of multiple special staining of bone tissue. DETAILED DESCRIPTION

[0052] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0053] In the following examples, rat knee joints are used as samples, but it should be noted that the method of the present invention is designed based on the common characteristics of bone tissue and is applicable to all calcium-containing hard tissues, and is not dependent on specific animal species or joint parts. Although the results only show the staining effect and analysis results using rat knee joints as an example, the method is also applicable to other types of bone tissue samples;

[0054] In the following examples, the reagents involved are:

[0055] (1) Nuclear dye: Weigert's iron hematoxylin;

[0056] (2) Cartilage dye: 5% Alcian blue (pH 2.5);

[0057] (3) Bone dye: 5% naphthol green;

[0058] (4) Collagen dye: 0.05% Sirius red;

[0059] (5) Washing solution 1: 3% acetic acid;

[0060] (6) Washing solution 2: 1% acetic acid.

[0061] Example 1 Reagent preparation and sample grouping

[0062] 1. Reagent configuration:

[0063] 1. Nuclear dye: Mix equal volumes of 1% hematoxylin solution (prepared with anhydrous ethanol) and 8% iron alum solution (prepared with distilled water). The mixed dye is Weigert's iron hematoxylin dye, which needs to be prepared and used immediately.

[0064] 2. Cartilage dye: Dissolve 5g of Alcian blue powder in 100ml of distilled water, adjust the pH value of the solution to 2.5 with acetic acid, mix well, and filter with filter paper before use;

[0065] 3. Bone dye: Dissolve 5g of naphthol green powder in 100ml of distilled water, mix well, and filter with filter paper before use;

[0066] 4. Collagen dye: Add 50 mg of Sirius red powder to 100 ml of saturated picric acid solution, mix well, and filter with filter paper before use;

[0067] 5. Differentiation solution: Mix 30 ml of distilled water and 70 ml of anhydrous ethanol to make 30% ethanol, take 99 ml, add 1 ml of concentrated hydrochloric acid and mix well;

[0068] 6. Blueing solution: add 1 ml of concentrated ammonia to 99 ml of distilled water and mix well;

[0069] 7. 3% acetic acid: add 3 ml of acetic acid to 97 ml of distilled water and mix well;

[0070] 8. 1% acetic acid: Add 1 ml of acetic acid to 99 ml of distilled water and mix well.

[0071] 2. Sample grouping and decalcification

[0072] 1. Number of samples: N = 5, total number of samples: N = 5 * 3 groups = 15

[0073] 2. Sample grouping: moderate decalcification (n days, moderate decalcification time, the specific number of days depends on the actual experimental conditions), slightly over-decalcification with acid (2n days, slightly extended decalcification time, used to simulate slightly over-decalcification), and over-decalcification with acid (4n days, significantly extended decalcification time, used to study the effects of over-decalcification). Where n is the decalcification time, and in the present invention, n=14 days.

[0074] 3. Taking 10% formic acid decalcification solution as an example, the decalcification steps are briefly described:

[0075] (1) Sample collection

[0076] Before decalcification, the rat knee joint samples were placed in 10% neutral formaldehyde solution and stored at 4°C for 24 hours to maintain the integrity of tissue structure and morphology.

[0077] (2) Decalcification

[0078] The samples were immersed in freshly prepared 10% formic acid decalcification solution, the solution volume was about 10 times the volume of the tissue, and decalcification was performed at room temperature (25°C). Ensure that the sample is completely immersed in the decalcification solution, place it on a vertical shaker and shake it slowly to promote decalcification efficiency, check the hardness change of the sample every two days, evaluate the decalcification progress by physical methods (such as needle puncture test), and replace it with freshly prepared 10% formic acid decalcification solution until the predetermined decalcification state is reached.

[0079] (3) Termination of decalcification

[0080] When the predetermined decalcification state and the preset decalcification time are reached, the sample is immediately removed from the decalcification solution and washed with distilled water overnight to remove residual formic acid. The washed sample is then subjected to subsequent tissue dehydration, paraffin embedding, paraffin sectioning, histological staining and other experiments.

[0081] Example 2 Development of staining methods (i) Preparation of decalcified rat knee bone samples and sample evaluation, staining with traditional techniques;

[0082] 1. Sample evaluation

[0083] After the decalcified samples are prepared, HE staining and traditional bone tissue staining techniques are used to evaluate the samples and determine the degree of decalcification. HE staining is one of the most commonly used staining techniques in pathology, used to observe the cell nucleus and cytoplasm in tissue sections. The cell nucleus will be stained blue and the cytoplasm will be stained pink. The cell nucleus of the acid-decalcified sample is often not colored or pink because the physical and chemical properties and acid-base environment are destroyed. Figure 1 It can be seen that the cell nuclei of the samples in the moderately decalcified and slightly over-decalcified acid groups are blue, and the coloring of the moderately decalcified group is stronger than that of the slightly over-decalcified acid group. However, the cell nuclei of the over-decalcified acid group are not colored or are red. This shows that the prepared samples meet the experimental requirements.

[0084] 2. Traditional bone tissue staining techniques (toluidine blue staining and safranin O fast green staining)

[0085] Depend on Figure 2-3 It can be seen that the cartilage and bone in the samples of the moderately decalcified group and the slightly over-decalcified acid group are brightly stained and well distinguished. The staining of the moderately decalcified group is stronger than that of the slightly over-decalcified acid group. The cartilage in the over-decalcified acid group is lightly stained or not stained. This shows that the prepared samples meet the experimental requirements, and at the same time, it can be seen that the limitations of the application of traditional staining technology in samples with different degrees of decalcification can be seen.

[0086] (ii) Single dyeing with dye solution

[0087] The main purpose of single dyeing is to optimize the experimental parameters. Use three dyes, keep the dyeing time unchanged under the corresponding conditions, adjust the concentration for single dyeing, evaluate the dyeing results, and determine the appropriate conditions for the dye. Figure 4-8This is the result of single staining. You can see gray-black cell nuclei, blue cartilage, green bone, the layered structure of the bone matrix is ​​also very clear, as well as red collagen. Different types of collagen can also be distinguished under a polarized light microscope.

[0088] (III) Double dyeing with dye solution

[0089] The four dye solutions were paired in pairs for double dyeing, with a focus on the mutual covering between the dye solutions. Figure 9-14 For double staining results, each dye solution is better with Weigert's iron hematoxylin, which will dye the cell nucleus gray-black, which is not easy to fade and easier to distinguish by color. The three sets of dye solutions, cartilage and bone, cartilage and collagen, and bone and collagen, have good differentiation and bright colors. The boundaries, morphology, and internal structure of cartilage and bone can be clearly distinguished, the distribution and functional relationship of collagen in cartilage can be understood, and the direction of collagen fibers in bone and their relationship with other components of bone can be understood.

[0090] (IV) Multiple special staining of bone tissue

[0091] Based on the above research, according to the chemical properties of the four dyes, their affinity for bone tissue components and the staining reaction mechanism, combined with the experimental conditions determined in the previous single staining and double staining, the staining sequence was arranged, and appropriate treatment was performed before and after staining with each dye to ensure that each dye was independent of each other and did not affect each other, thus designing a bone tissue multiple special staining method. The specific operation is as follows:

[0092] 1. Bone tissue samples are decalcified, embedded in paraffin, and sectioned;

[0093] 2. Dewaxing and hydration of paraffin sections: immerse the paraffin sections in xylene substitute for 15 minutes × 3 times, anhydrous ethanol for 5 minutes × 2 times, 95% ethanol for 5 minutes × 2 times, and 80% ethanol for 5 minutes. Soak in distilled water for 2 minutes;

[0094] 3. Wash with 3% acetic acid for 3 min;

[0095] 4. Cartilage staining: stain with 5% Alcian blue solution (pH 2.5) for 20 min, and wash with 1% acetic acid;

[0096] 5. Nucleus staining: Weigert's iron hematoxylin staining for 10 minutes, wash with tap water, add differentiation solution for differentiation, and turn blue; 6. Bone staining: 5% naphthol green staining for 3 minutes, wash with 1% acetic acid;

[0097] 7. Collagen staining: stain with 0.05% Sirius red solution for 8 minutes, and wash with 1% acetic acid;

[0098] 8. Dehydration and transparency of sections: immerse the paraffin sections in 95% ethanol for 2 min × 2 times, anhydrous ethanol for 2 min × 2 times, xylene or xylene substitute for 5 min × 2 times;

[0099] 9. Seal the film with neutral gum;

[0100] 10. Image acquisition: Wait until the neutral gum is completely dry before image acquisition can be carried out, which should be completed within one month;

[0101] 11. Slice storage: After the slices are placed in the slice box, they should be kept away from light and at room temperature;

[0102] 12. Spectral splitting and data analysis: Use inform analysis software to extract the spectrum of the dye solution single dyeing results, and perform subsequent data analysis based on the experimental purpose.

[0103] The results are as follows Figure 15-16 As shown in the figure, under this multiple special staining scheme, the staining and differentiation of four tissue structures, namely, cell nuclei, cartilage, bone and collagen in bone tissue can be achieved simultaneously, specifically:

[0104] Cell nucleus: gray-black, gray-blue, no staining (acid over-decalcification group);

[0105] Cartilage: blue, cyan;

[0106] Hard bone: green, yellow-green;

[0107] Collagen: red.

[0108] Different collagen types can be observed under a polarized light microscope. Different collagen types can be observed under a polarized light microscope. An optimized multiple special staining scheme for bone tissue was successfully established.

[0109] Example 3 Analysis of bone tissue staining results

[0110] Based on the principle of spectroscopy, the inform software can accurately capture the characteristic spectrum of the sample tissue structure through spectral extraction, and use the split analysis function to decompose the mixed spectral information according to the spectral database and recognition model. It can accurately identify and clearly distinguish the sample tissue structure, and also has powerful statistical analysis functions. The software was used to analyze the stained sample slices of Example 2:

[0111] 1. Spectral information collection

[0112] (1) Spectral scanning: Place the bone tissue slices stained with dye solution in the PhenoImager HT fully automatic multispectral tissue slice imaging system, and use a high-resolution spectral camera to capture the spectral information of each dye and each stained area. Each dye has its own unique absorption and reflection characteristics, which are manifested as a specific wavelength distribution on the spectrum.

[0113] (2) Spectral extraction: Based on the principle of spectroscopy, the inform software can accurately capture the characteristic spectrum of each dye. The built-in algorithm of the software will automatically identify and extract the specific spectral characteristics of each dye and establish the corresponding spectral database ( Fig.17 ), which is an important reference for the subsequent separation of mixed spectra.

[0114] 2. Spectral splitting

[0115] The double-stained and multi-stained bone tissue sections were imaged in the same way. The split analysis function of the inform software was used to decompose the mixed spectral information into the spectral characteristics of each component based on the pre-established spectral database and the recognition model built into the software, thereby separating the staining results of a single dye from the mixed staining ( Figure 18-24 ). This process ensures that the spectral features of structures labeled with different dyes are clearly presented after separation and do not interfere with each other, proving the effectiveness of the multiple staining method.

[0116] 3. Tissue structure identification and statistical analysis

[0117] (1) Accurate identification: Through the above spectral decomposition steps, the inform software can accurately identify different tissue structures in the sample, thereby achieving clear morphological distinction.

[0118] (2) Statistical analysis: The software also provides powerful statistical analysis functions. Based on the research purpose, the distribution, relative proportions, and spatial relationships of different staining components can be further quantitatively analyzed to achieve a more comprehensive understanding of the complex structure and composition changes of bone tissue.

[0119] Fig.17 It is the spectral information of the single staining results of the four dye solutions, which can be used for subsequent double / multiple staining result signal separation. Figure 18-24 To separate the results of double and multiple dye staining, the spectral features of the four dye-labeled structures are clearly presented after separation, can be accurately extracted and do not interfere with each other.

[0120] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A bone tissue multiple special staining analysis method, characterized in that: The method comprises the following steps: (1) Cartilage staining: staining with 5% Alcian blue solution (pH 2.5) for 20 min, followed by washing with 1% acetic acid; (2) Nuclear staining: Weigert's iron hematoxylin staining for 10 min, washing with tap water, differentiation, and blueing; (3) Bone staining: 5% naphthol green staining for 3 min, washed with 1% acetic acid; (4) Collagen staining: staining with 0.05% Sirius red solution for 8 min, followed by washing with 1% acetic acid.

2. The method according to claim 1, characterized in that The method also includes pre-staining steps such as decalcification of bone tissue samples, paraffin embedding, paraffin sectioning, section dewaxing and hydration, and acetic acid washing.

3. The method according to claim 2, characterized in that The decalcification method is to use 10% formic acid for decalcification.

4. The method according to claim 1, characterized in that The method further comprises dehydrating the slices and sealing the slices with neutral gum after staining.

5. The method according to any one of claims 1 to 4, characterized in that: The method also includes using inform analysis software to extract the spectrum of the staining results and perform data analysis according to the experimental purpose.

6. A bone tissue multiple special staining analysis kit, characterized in that: The kit includes 5% Alcian blue solution, 1% acetic acid, Weigert's iron hematoxylin, 5% naphthol green solution, and 0.05% Sirius red solution.

7. The bone tissue multiple special staining analysis kit according to claim 6, characterized in that: The kit is used according to the analysis method described in any one of claims 1 to 5.

8. Use of the analysis method according to any one of claims 1 to 5 in analyzing the morphological structure of bone tissue.

9. Use of the kit as claimed in claim 6 or 7 in analyzing the morphological structure of bone tissue.

10. The use according to claim 9, characterized in that The kit is used in conjunction with inform analysis software.

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