Preparation method of paraffin embedded section of decalcified bone tissue

Abstract

The invention provides a preparation method of a paraffin-embedded section of decalcified bone tissue, which belongs to the field of paraffin section manufacturing and comprises the steps of fixation, decalcification, gradient concentration ethanol treatment, embedding, freezing, slicing and the like. The method for treating the glass slide is simple and low in cost, a uniform film can be formed, the section keeps good adhesiveness in the embedding and dyeing process, meanwhile, the solvent resistance is good, the problem of section falling caused by xylene dewaxing is solved, tissue loss can be remarkably reduced, and the experiment stability and repeatability are improved. In addition, a temperature gradient wax dipping method is also used, so that the permeation rate of paraffin inside and on the surface of the tissue is more uniform, a uniform and stable paraffin embedding matrix is finally formed, the slice quality is improved, and the dyeing and microscopic observation effects are clearer.

Description

Technical Field

[0001] The invention belongs to the field of paraffin section production, and in particular relates to a method for preparing paraffin-embedded sections of decalcified bone tissue. Background Art

[0002] Bone tissue pathological analysis is a core tool for diagnosing metabolic bone diseases, bone tumors, and assessing bone repair status. Bone biopsy, the gold standard for diagnosing metabolic bone diseases, bone tumors, and assessing bone metabolism, directly determines the accuracy of clinical diagnosis. The iliac bone, the most active anatomical site for bone remodeling, exhibits a heterogeneous structure of both cortical and cancellous bone in its biopsy specimens, providing a critical window into the dynamics of the bone microenvironment. However, traditional bone tissue preparation techniques are plagued by bottlenecks such as limited human tissue experimental data, subjective experience in determining decalcification endpoints, and high rates of section delamination. This leads to poor staining quality and frequent tissue tears, severely interfering with quantitative analysis of human bone quality and molecular marker detection. With the stringent requirements of precision medicine for the reproducibility of pathological data and the reliance of bone-targeted drug development on high-resolution bone morphology, innovative bone tissue decalcification and preparation technologies are urgently needed to improve diagnostic consistency and unlock molecular insights into bone microarchitecture. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a method for preparing paraffin-embedded sections of decalcified bone tissue, to improve the current preparation process of decalcified paraffin-embedded human bone tissue, and to overcome the shortcomings of the preparation process being complicated, the end point of iliac bone decalcification being unclear, and the sectioning being detached during the staining process.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for preparing paraffin-embedded sections of decalcified bone tissue, comprising the following steps:

[0006] 1) Place bone tissue in a fixative at 0-6°C for 48-96 hours, then wash away the fixative to obtain the fixed bone tissue.

[0007] 2) decalcifying the fixed bone tissue with a decalcifying solution, and then washing away the decalcifying solution to obtain decalcified bone tissue;

[0008] 3) treating the decalcified bone tissue with ethanol solutions of different volume concentrations in sequence, treating the decalcified bone tissue with an ethanol solution of xylene for 3 to 10 minutes, and then treating the decalcified bone tissue in xylene for 20 to 40 minutes to obtain treated bone tissue;

[0009] 4) embedding the processed bone tissue in paraffin, freezing it, and slicing it to obtain bone tissue sections;

[0010] 5) Spreading the bone tissue slices, mounting them on a sheet, and drying them at 50-70°C for 90-150 minutes to obtain paraffin-embedded sections of decalcified bone tissue;

[0011] Step 3) The treatment method of the different solutions is: 60% to 75% ethanol solution treatment for 20 to 40 minutes, 70% to 80% ethanol solution treatment for 20 to 40 minutes, 75% to 85% ethanol solution treatment for 20 to 40 minutes, 80% to 90% ethanol solution treatment for 40 to 80 minutes, 90% to 100% ethanol solution treatment for 40 to 80 minutes, 95% to 100% ethanol solution treatment for 40 to 80 minutes, and 95% to 100% ethanol solution treatment for 1 to 2 hours;

[0012] The embedding method is as follows: immersing in paraffin at 54-58° C. for 40-80 minutes, then immersing in paraffin at 56-60° C. for 90-150 minutes, and then immersing in paraffin at 63-70° C. for 2-4 hours.

[0013] Preferably, the fixative in step 1) is 2% to 6% paraformaldehyde.

[0014] Preferably, the bone tissue collected in step 1) has a diameter of 2 to 5 mm and a length of 0.5 to 2 cm.

[0015] Preferably, the decalcification solution in step 2) is a 0.3-1 mmol / L EDTA-2Na solution.

[0016] Preferably, the volume ratio of ethanol to xylene in the xylene ethanol solution in step 3) is 1-3:1-3.

[0017] Preferably, the thickness of the bone tissue slice in step 4) is 2 to 6 μm.

[0018] Preferably, in step 5), the bone tissue slices are unfolded in water at 40-50°C.

[0019] Preferably, in step 5), the patch is formed by placing the bone tissue slice after spreading on a treated glass slide;

[0020] The processing method of the slide is as follows: immersing the slide in a solution containing 0.5% to 1% gelatin and 0.02% to 0.1% potassium chromium sulfate at 50 to 70° C., leaving it to stand for 8 to 12 minutes, and then drying it at 35 to 40° C. for 3 to 5 hours.

[0021] Preferably, the bone tissue includes ilium.

[0022] The present invention provides paraffin-embedded sections of decalcified bone tissue prepared by the preparation method.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The method of the present invention uses EDTA decalcification, which can gently and slowly remove calcium ions from bone tissue, better preserve tissue morphology and its molecular integrity during the decalcification process, effectively preserve the antigenicity of proteins, and is compatible with subsequent immunohistochemistry, in situ hybridization, fluorescence microscopy and molecular biology detection, expanding its application range in scientific research and clinical pathology. In addition, the present invention uses gelatin coating to prevent detachment, which is inexpensive to prepare and has strong operability. Compared with commercially available polylysine slides, which have insufficient adhesion on high-hardness sections of bone tissue and during xylene dewaxing (bone slice detachment rate ≥80%), APES silane coating is solvent-resistant but expensive. The gelatin of the present invention can form a uniform film, so that the slices maintain good adhesion during paraffin embedding and subsequent staining, and have good solvent resistance, avoiding the problem of slice detachment caused by xylene dewaxing. It can significantly reduce tissue loss, improve experimental stability and repeatability, and is particularly suitable for cost-sensitive laboratories and large-scale tissue sample analysis needs. The present invention also uses a temperature gradient wax infiltration method to achieve a more uniform paraffin penetration rate within and on the tissue surface, ultimately forming a uniform and stable paraffin embedding matrix, improving section quality and enabling clearer staining and microscopic observation. This avoids the "false embedding" phenomenon in traditional embedding methods, where the tissue surface rapidly solidifies while the dehydrating agent remains inside due to the different solubility of the dehydrating agent and paraffin, which can easily cause trabeculae to fall off or break during sectioning. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Paraffin-embedded sections of decalcified bone tissue obtained by the preparation method of Comparative Example 1;

[0026] Figure 2 Paraffin-embedded sections of decalcified bone tissue prepared in Example 1;

[0027] Figure 3 This is the HE staining result of human iliac bone biopsy tissue prepared in Example 1;

[0028] Figure 4 This is the HE staining result of human iliac bone biopsy tissue prepared from untreated slides. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing paraffin-embedded sections of decalcified bone tissue, comprising the following steps:

[0030] 1) Place bone tissue in a fixative at 0-6°C for 48-96 hours, then wash away the fixative to obtain the fixed bone tissue.

[0031] 2) decalcifying the fixed bone tissue with a decalcifying solution, and then washing away the decalcifying solution to obtain decalcified bone tissue;

[0032] 3) treating the decalcified bone tissue with ethanol solutions of different volume concentrations in sequence, treating the decalcified bone tissue with an ethanol solution of xylene for 3 to 10 minutes, and then treating the decalcified bone tissue in xylene for 20 to 40 minutes to obtain treated bone tissue;

[0033] 4) embedding the processed bone tissue in paraffin, freezing it, and slicing it to obtain bone tissue sections;

[0034] 5) Spreading the bone tissue slices, mounting them on a sheet, and drying them at 50-70°C for 90-150 minutes to obtain paraffin-embedded sections of decalcified bone tissue;

[0035] In the present invention, bone tissue is placed in a fixative, fixed at 0-6°C for 48-96 hours, and then the fixative is washed off to obtain the fixed bone tissue. In the present invention, the bone tissue is preferably collected by inserting a bone biopsy needle into the flat bone surface 1-2 cm behind the anterior superior iliac spine, and the collected length is preferably 0.5-2 cm, more preferably 0.7-1.5 cm, and even more preferably 1 cm; the collected diameter is preferably 1-5 mm, more preferably 2-4 mm, and even more preferably 3 mm; the fixative used for the fixation is paraformaldehyde, and the concentration of paraformaldehyde is 2%-6%, preferably 3%-5%, and even more preferably 4%. The fixation temperature is 0-6°C, preferably 2-5°C, and more preferably 4°C. The volume of the fixative used is preferably 30-70 times the volume of the bone tissue, more preferably 40-60 times, and more preferably 50 times; the fixation time is 48-96h, preferably 60-84h, and more preferably 72h; the method for washing away the fixative is preferably rinsing with clean water, and the rinsing time is preferably 0.5-2h, more preferably 0.7-1.5h, and more preferably 1h.

[0036] In the present invention, the fixed bone tissue is decalcified with a decalcifying solution, and the decalcifying solution is washed away to obtain decalcified bone tissue. In the present invention, the decalcifying solution is preferably an EDTA-2Na solution, and the decalcification method is preferably to wash the fixed bone tissue from the fixative and then immerse it in the decalcifying solution. The volume of the decalcifying solution used is preferably 30 to 70 times the volume of the bone tissue, more preferably 40 to 60 times, and even more preferably 50 times. The concentration of the EDTA-2Na solution is preferably 0.3 to 1 mmol / L, more preferably 0.4 to 0.7 mmol / L, and even more preferably 0.5 mmol / L. The decalcification time is preferably 0.5 to 3 months, more preferably 0.7 to 2 months, and even more preferably 1 month. The decalcifying solution is preferably replaced every 1 to 5 days, more preferably 2 to 4 days, and even more preferably 3 days. The decalcification process is completed until the insulin needle penetrates the cortical bone end along the longitudinal axis without resistance; the decalcification solution is preferably washed away with clean water, and the washing time is preferably 0.5 to 2 hours, more preferably 0.7 to 1.5 hours, and even more preferably 1 hour.

[0037] In the present invention, the decalcified bone tissue is treated with ethanol solutions of different volume concentrations in sequence, and the treatment method is preferably soaking. After being treated with the ethanol solution of xylene for 3 to 10 minutes, the bone tissue is placed in xylene for treatment for 20 to 40 minutes to obtain the treated bone tissue.

[0038] The treatment method of the different solutions is as follows: the volume concentration of the ethanol solution is 60% to 75%, preferably 65% ​​to 72%, more preferably 70%, and the treatment time is 20 to 40 minutes, preferably 25 to 35 minutes, more preferably 30 minutes;

[0039] The volume concentration of the ethanol solution is 70% to 80%, preferably 72% to 78%, more preferably 75%, and the treatment time is 20 to 40 minutes, preferably 25 to 35 minutes, more preferably 30 minutes;

[0040] The volume concentration of the ethanol solution is 75% to 85%, preferably 78% to 82%, more preferably 80%, and the treatment time is 20 to 40 minutes, preferably 25 to 35 minutes, more preferably 30 minutes;

[0041] The volume concentration of the ethanol solution is 80% to 90%, preferably 82% to 88%, more preferably 85%, and the treatment time is 40 to 80 minutes, preferably 50 to 70 minutes, more preferably 60 minutes;

[0042] The volume concentration of the ethanol solution is 90% to 100%, preferably 92% to 98%, more preferably 95%, and the treatment is 40 to 80 minutes, preferably 50 to 70 minutes, more preferably 60 minutes;

[0043] The volume concentration of the ethanol solution is 95% to 100%, preferably 100%, and the treatment time is 40 to 80 minutes, preferably 50 to 70 minutes, and more preferably 60 minutes;

[0044] The volume concentration of the ethanol solution is 95% to 100%, preferably 100%, and the treatment time is 1 to 2 hours, preferably 1.2 to 1.8 hours, and more preferably 1.5 hours;

[0045] The xylene ethanol solution is treated for 3 to 10 minutes, preferably 4 to 8 minutes, and more preferably 5 minutes. The volume ratio of xylene to ethanol in the xylene ethanol solution is 1 to 3:1 to 3, preferably 1:1;

[0046] The xylene treatment time is 40 to 80 minutes, preferably 50 to 70 minutes, and more preferably 60 minutes.

[0047] In the present invention, the ethanol concentration is increased gradually by 5% in the early stages, resulting in a more gradual change. This prevents tissue shrinkage or microstructural damage caused by drastic changes in osmotic pressure, making it particularly suitable for brittle, hardened tissues such as decalcified bone. Later, by extending the anhydrous ethanol treatment time and repeating the treatment once, residual moisture is thoroughly removed, preventing subsequent emulsification of the clearing agent with water, which could affect the clearing effect and paraffin penetration. Furthermore, using an equal proportion of anhydrous ethanol and xylene as a transitional step mitigates the dramatic tissue shrinkage caused by the polarity difference between the ethanol and xylene when they come into direct contact, ensuring a smoother clearing process.

[0048] This method uses ethanol as a dehydrating agent, achieving gradient dehydration by displacing water within the tissue, thus preventing structural changes caused by rapid water loss. Xylene is also used as a clearing agent. Xylene rapidly dissolves ethanol and displaces lipids within the tissue, making it transparent and facilitating uniform paraffin wax penetration, significantly shortening the clearing time. Compared to other clearing agents, such as mixed solvents like tert-butyl alcohol, acetone, and n-butanol, xylene is inexpensive, and its use alone simplifies the process and reduces reagent management complexity.

[0049] The volumes of the different solutions used are preferably 10 to 30 times that of the bone tissue, more preferably 15 to 25 times, and even more preferably 20 times.

[0050] The present invention embeds the processed bone tissue in paraffin, freezes and slices it to obtain bone tissue slices; the embedding method is: immersing in paraffin at 54-58°C, preferably 55-57°C, more preferably 55°C, for 40-80 minutes, preferably 50-70 minutes, more preferably 60 minutes; immersing in paraffin at 56-60°C, preferably 57-59°C, more preferably 58°C, for 90-150 minutes, preferably 100-130 minutes, more preferably 120 minutes; and then immersing in paraffin at 63-70°C, preferably 64-68°C, more preferably 65°C, for 2-4 hours, preferably 2.5-3.5 hours, more preferably 3 hours.

[0051] In the present invention, the freezing temperature of the bone tissue is preferably -30 to -10°C, more preferably -25 to -15°C, and even more preferably -20°C; the freezing time is preferably 8 to 16 hours, more preferably 10 to 14 hours, and even more preferably 12 hours; the thickness of the bone tissue slice is preferably 2 to 6 μm, more preferably 3 to 5 μm, and even more preferably 4 μm.

[0052] In the present invention, the bone tissue slices are spread and then applied to a sheet, and then dried at 50-70°C for 90-150 minutes to obtain paraffin-embedded sections of decalcified bone tissue. The spread is preferably developed in water at 40-50°C, more preferably 41-45°C, and even more preferably 42°C. The applied section is preferably placed on a treated glass slide after the spread. The processing method of the slide is as follows: immersing the slide in a solution containing gelatin and potassium chromium sulfate, standing and then drying; the temperature of the solution is preferably 50-70°C, more preferably 55-65°C, and more preferably 60°C; the mass concentration of gelatin is preferably 0.5%-1%, more preferably 0.6%-0.8%, and more preferably 0.7%; the mass concentration of potassium chromium sulfate is preferably 0.02%-0.1%; the standing time is preferably 8-12 minutes, more preferably 9-11 minutes, and more preferably 10 minutes; the drying temperature is preferably 35-40°C, more preferably 36-38°C, and more preferably 37°C; the drying time is preferably 3-5 hours, more preferably 3.5-4.5 hours, and more preferably 4 hours. The present invention uses potassium chromium sulfate as a cross-linking agent, through its metal ion (Cr 3+ ) forms coordination bonds with the carboxyl or amino groups of gelatin, strengthening the network structure of gelatin. It is also thermally stable and can maintain adhesion during the subsequent long-term antigen heat repair process, maintaining the adsorption of the slide and tissue.

[0053] In the present invention, the drying temperature is 50-70° C., preferably 55-65° C., more preferably 60° C., and the drying time is 90-150 min, preferably 100-130 min, more preferably 120 min.

[0054] The present invention provides paraffin-embedded sections of decalcified bone tissue prepared by the preparation method.

[0055] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1 Preparation of HE staining of human iliac bone biopsy tissue

[0057] A bone biopsy needle was inserted into the flat bone surface 1-2 cm posterior to the patient's anterior superior iliac spine to obtain a 3 mm diameter and 1 cm long sample. The sample was fixed in 4% paraformaldehyde at 4°C for 72 hours. Excess soft tissue was removed from the sample, and the sample was rinsed with clean water for 1 hour. After removing the fixative, the fixed bone tissue was obtained.

[0058] Use phosphate buffer to prepare 0.5mmol / L EDTA-2Na to prepare decalcification solution, and use 1mmol / L NaOH to adjust the pH value to 7.0-7.2. Use the prepared decalcification solution to immerse the fixed bone tissue for decalcification at room temperature. Change the decalcification solution every three days for 1 month. Use an insulin needle to pierce the cortical bone end along the longitudinal axis. If there is no resistance, it means decalcification is complete. Rinse with clean water for 1 hour, remove the decalcification solution, and obtain decalcified bone tissue.

[0059] The decalcified bone tissue was treated with different solutions: immersion in 70% ethanol (20 times the volume of decalcified bone tissue) for 30 minutes, 75% ethanol (20 times the volume of decalcified bone tissue) for 30 minutes, 80% ethanol (20 times the volume of decalcified bone tissue) for 30 minutes, 85% ethanol (20 times the volume of decalcified bone tissue) for 1 hour, 95% ethanol (20 times the volume of decalcified bone tissue) for 1 hour, anhydrous ethanol (20 times the volume of decalcified bone tissue) for 1 hour, and then re-treatment for 1.5 hours after replacing anhydrous ethanol. Then, ethanol solution (anhydrous ethanol and xylene mixed in equal proportions) of 20 times the volume of decalcified bone tissue was treated for 5 minutes, and the bone tissue was placed in a tissue embedding box. After treatment with xylene for 30 minutes, xylene was replaced and treated again for 30 minutes to obtain the treated tissue embedding box.

[0060] The treated tissue embedding cassette was immersed in a wax bath with a temperature gradient for 6 hours, and then placed in a -20°C freezer overnight. Embedding was performed using a PBM-A pathology tissue freezing and embedding station.

[0061] The temperature gradient paraffin immersion lasted for 6 hours, specifically: immersing the tissue embedding box in 56°C paraffin for 1 hour, immersing the tissue embedding box in 58°C paraffin for 2 hours, and immersing the tissue embedding box in 65°C paraffin for 3 hours.

[0062] Paraffin sections were made using a HistoCore microtome (Leica) using High-Profile Disposable Blades 818 (Leica). After trimming to the optimal position, sections were cut to a thickness of 3 to 5 μm. After observing the integrity of the section, the sections were unfolded in a 42°C waterbath. The sections were then removed using a treated glass slide and dried in an oven at 60°C for 2 hours to obtain paraffin-embedded sections of decalcified bone tissue. For details, see [ 15 ] Figure 2 It can be used for HE staining, immunohistochemistry staining, immunofluorescence staining, alkaline phosphatase staining or tartrate-resistant acid phosphatase (TRAP) staining, etc.

[0063] The treated glass slides were prepared by heating deionized water containing 0.7% gelatin and 0.05% potassium chromium sulfate at 60°C until no crystalline particles remained. The glass slides were vertically immersed in the 60°C gelatin solution and allowed to stand for 10 minutes. The coated glass slides were removed and dried in a 37°C oven for 4 hours.

[0064] The prepared paraffin-embedded sections of decalcified bone tissue were immersed in xylene for 5 minutes, then replaced with xylene for 5 minutes, then replaced with anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, and 75% ethanol for 5 minutes, and then gently rinsed with clean water 3 times until the water flowed down naturally without sticking to the wall.

[0065] Soak in 0.5% hematoxylin solution for 2 minutes, rinse gently with water 3 times, immerse in differentiation solution (1% hydrochloric acid ethanol solution) for 1 second, rinse gently with water again, and soak in deionized water for 20 minutes.

[0066] The sections were stained with 0.5% eosin solution (alcohol-soluble) for 1 minute, immersed in 95% ethanol for 1 minute, and immersed in absolute ethanol for 1 minute.

[0067] Air-dry, seal with neutral gum, and obtain human iliac bone biopsy tissue for HE staining. Figure 3 .

[0068] Comparative Example 1

[0069] Dehydrate for 1 hour in each gradient using 70% ethanol solution, 80% ethanol solution, 90% ethanol solution, 100% ethanol and tert-butanol mixture, 100% ethanol and acetone mixture, and 100% n-butanol solution. Transparent treatment was performed using chloroform-anhydrous ethanol 5:1 for 8 hours, and then replaced with chloroform for 24 hours (Patent No. CN201810872614.7). For details on sections, see Figure 1 ; Figure 1It shows that there is a large amount of separation within the tissue block, poor dehydration, and poor tissue integrity.

[0070] Comparative Example 2 HE staining of human iliac bone biopsy tissue prepared using untreated slides

[0071] The treated glass slide in Example 1 was replaced with an untreated glass slide, and the other steps were the same as in Example 1 to obtain human iliac bone biopsy tissue HE staining. The results are shown in detail. Figure 4 , Figure 4 It showed that most of the trabeculae were detached from the slide.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing paraffin-embedded sections of decalcified bone tissue, characterized in that: The following steps are involved: 1) Place bone tissue in a fixative at 0-6°C for 48-96 hours, then wash away the fixative to obtain the fixed bone tissue. 2) decalcifying the fixed bone tissue with a decalcifying solution, and then washing away the decalcifying solution to obtain decalcified bone tissue; 3) treating the decalcified bone tissue with different solutions in sequence, treating the decalcified bone tissue with a xylene ethanol solution for 3 to 10 minutes, and then treating the decalcified bone tissue in xylene for 20 to 40 minutes to obtain treated bone tissue; 4) embedding the processed bone tissue in paraffin, freezing it, and slicing it to obtain bone tissue sections; 5) Spreading the bone tissue slices, mounting them on a sheet, and drying them at 50-70°C for 90-150 minutes to obtain paraffin-embedded sections of decalcified bone tissue; Step 3) The treatment method of the different solutions is: 60% to 75% ethanol solution treatment for 20 to 40 minutes, 70% to 80% ethanol solution treatment for 20 to 40 minutes, 75% to 85% ethanol solution treatment for 20 to 40 minutes, 80% to 90% ethanol solution treatment for 40 to 80 minutes, 90% to 100% ethanol solution treatment for 40 to 80 minutes, 95% to 100% ethanol solution treatment for 40 to 80 minutes, and 95% to 100% ethanol solution treatment for 1 to 2 hours; The embedding method is as follows: immersing in paraffin at 54-58° C. for 40-80 minutes, then immersing in paraffin at 56-60° C. for 90-150 minutes, and then immersing in paraffin at 63-70° C. for 2-4 hours.

2. The preparation method according to claim 1, characterized in that The fixing solution in step 1) is 2% to 6% paraformaldehyde.

3. The preparation method according to claim 1, characterized in that In step 1), the bone tissue is collected with a diameter of 2 to 5 mm and a length of 0.5 to 2 cm.

4. The preparation method according to claim 1, characterized in that Step 2) The decalcification solution is a 0.3-1 mmol / L EDTA-2Na solution.

5. The preparation method according to claim 1, characterized in that In step 3), the volume ratio of ethanol to xylene in the xylene ethanol solution is 1-3:1-3.

6. The preparation method according to claim 1, characterized in that Step 4) The thickness of the bone tissue slices is 2 to 6 μm.

7. The preparation method according to claim 1, characterized in that Step 5) The bone tissue slices are unfolded in water at 40-50°C.

8. The preparation method according to claim 1, characterized in that Step 5) The patch is formed by placing the bone tissue slice after spreading on a treated glass slide; The processing method of the slide is as follows: immersing the slide in a solution containing 0.5% to 1% gelatin and 0.02% to 0.1% potassium chromium sulfate at 50 to 70° C., leaving it to stand for 8 to 12 minutes, and then drying it at 35 to 40° C. for 3 to 5 hours.

9. The preparation method according to claim 1, characterized in that The bone tissue includes the ilium.

10. Paraffin-embedded sections of decalcified bone tissue prepared by the preparation method according to any one of claims 1 to 9.

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