Dyeing method of bone cell indentation-tubule system and kit thereof

Abstract

The invention discloses a dyeing method and kit for an osteocyte indentation-tubule system, and the dyeing method comprises the following steps: (1) preparing (0.5-1) mol / L AgNO3 as a solution I, (2) preparing 0.05%-2% (w / v) of B-type gelatin and 0.05%-5% (v / v) of formic acid as a solution II; and (3) mixing the solution I and the solution II in a volume ratio of (4-1): 1, and dyeing. The optimized dyeing step shows clear and reliable dyeing mode and strength in different species and disease models, and compared with a traditional method, the method has the advantages that more bone cell indentation-tubule systems can be dyed, and the density of the bone cell indentation-tubule systems can be increased.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a staining method for a bone cell lacunae-tubule system and a kit thereof. Background Art

[0002] Osteocytes are differentiated, mature osteoblasts that reside in small holes in bones called lacunae. A notable feature of osteocytes is the large number of branch-like projections on the cell surface, called dendrites. These dendrites travel through a network of canaliculi, which are tubes in the bone matrix, and connect with other osteocyte dendrites. There are about 42 billion osteocytes in an adult skeleton, and each osteocyte has about 40-100 dendrites that extend into 20-126 surrounding canaliculi. Researchers call the network of holes and canaliculi around osteocytes and their dendrites the Osteocyte Lacuno-Canalicular System (LCS). The Osteocyte Lacuno-Canalicular System is essential for nutrient exchange, waste removal, and signal transduction between osteocytes and between osteocytes and other cells (such as osteoblasts, osteoclasts, vascular endothelial cells, etc.). Given that the osteocyte lacunae-canalicular system plays a vital role in maintaining bone physiological homeostasis and regulating bone remodeling and osteocyte mechanotransduction, many researchers have focused on its structural and functional changes in childhood bone development, bone diseases (such as osteoporosis, osteoarthritis, etc.), pregnancy, aging, and other physiological and pathological conditions.

[0003] In histological research, the protein silver method (Protargol-S or SilverProtein), ammonium silver method (silver nitrate and ammonium hydroxide), mercuric chloride method (HgCI2), thionin method (Thionin), Giemsa method and other methods have been used successively to stain the lacunar tubule system of osteocytes. Mercuric chloride method, thionin method and Giemsa method are no longer used due to unclear and unstable staining results. The ammonium silver method (silver nitrate and ammonium hydroxide) is no longer used in the field of bone biology research due to the complex solution preparation and debugging process and the unstable staining results. The protein silver method (Protargol-S or SilverProtein) is occasionally used to stain the lacunar tubule system of osteocytes, but due to the instability of the silver ion concentration contained in the protein silver dye, the complexity and time-consuming dye preparation and staining operation process, and the unclear staining results, the density of the stained lacunar tubule system is very low. At present, the protein silver method is rarely used to stain the lacunar tubule system of osteocytes.

[0004] In addition to the above abandoned methods, the method currently used for the histological analysis of the osteocyte lacunae-tubule system under various bone physiological and pathological conditions is mainly silver nitrate staining. However, this method has various problems. The variability of the concentrations of silver nitrate and gelatin-formic acid solution reported in the literature, the inconsistency of the staining procedures used, the variability of the type of gelatin used, and the variability of staining and termination conditions have limited the widespread application of this method in the field of osteocytes.

[0005] Specifically, the following aspects of the current silver nitrate staining protocol are unclear or variable: (1) the concentration of silver nitrate solution used by different researchers is inconsistent; (2) the variability of the concentration of the gelatin-formic acid combination solution; (3) the variability of the volume ratio of the silver nitrate solution to the gelatin-formic acid combination solution (4:1, 2:1, 1:1, 1:2 or 1:4, etc.); (4) the unclear type of gelatin used and the huge difference in the staining effect; (5) the unclear lighting conditions for staining (ultraviolet light, indoor white light, or dark); (6) the unclear conditions for stopping staining (deionized water, or 5% sodium thiosulfate solution); (7) the variability of tissue section types (paraffin sections, frozen sections).

[0006] The above variability factors in the existing staining schemes, the unclear reagent types and other defects have limited the widespread application and acceptance of this method in the field of bone cells, resulting in the great differences in the effects of the staining pictures reported in different literatures. Some researchers have clear contrast, high density and large number of osteocyte lacunae-tubule systems after staining, while others have unclear contrast, and the number and density are lower than the data of other researchers. In addition, some researchers' staining pictures have clear backgrounds, while some researchers' staining pictures have more or less black silver precipitate particles, resulting in inaccuracy in the quantitative analysis of osteocyte lacunae-tubule systems in specific species.

[0007] In addition, the high-concentration 50% silver nitrate solution may not fully present all the lacunae-tubule systems due to its strong reduction reaction, and it is also possible that the strong reduction reaction of the high-concentration silver nitrate reagent will damage the lacunae-tubule system. Therefore, when using existing methods to evaluate the number and density of lacunae-tubules in osteocytes in different species, there will be an underestimation, that is, the number of actual lacunae-tubules will be underestimated, which will lead to a deviation in the comparative study of the function and morphology of the lacunae-tubule systems of different species of animals.

[0008] At present, there is still no specific dye and staining kit for histological staining of the lacunae-tubule system of osteocytes on the market at home and abroad. The market reality of the lack of effective specific dyes and staining kits is in sharp contrast to the demand and research enthusiasm of clinicians and basic researchers for research methods on metabolic and immune bone diseases (such as osteoporosis, osteoarthritis, pediatric bone development disorders, rheumatic immune diseases, etc.) in the past 10 years. Therefore, the development of simpler, universal, effective, economical and environmentally friendly histological staining methods and the commercialization of corresponding specific dyes and staining kits will greatly promote the relevant research progress in the field of bone cell biology and provide a powerful tool for the research and clinical diagnosis of metabolic and immune bone diseases (such as osteoporosis, osteoarthritis, pediatric bone development disorders, rheumatic immune diseases, etc.) in children and adults. Summary of the invention

[0009] In order to solve the above technical problems, this patent systematically evaluates the various factors that affect the effect of the classic silver nitrate staining method, establishes a silver nitrate staining method and process that is more convenient, simple to operate, and more environmentally friendly than the traditional staining method and process, and prepares the corresponding staining solution into a freeze-dried powder dye to form a specific dye that can effectively stain the network of the osteocyte crypt-tubule system. Specifically, the present invention uses a (0.5-1) mol / L silver nitrate solution and a gelatin-formic acid combination solution of different concentration ranges (0.05%-0.5% type B gelatin and 0.05%-5% formic acid, or 1%-2% type B gelatin and 0.1%-2% formic acid), mixes them at a volume ratio of (4-1): 1, and then stains them under ultraviolet light in a biological safety cabinet for 60 minutes or under indoor white light or in the dark for 90 minutes. After staining, use Milli-Q pure water to wash away the staining solution directly, and then perform conventional dehydration, transparency and sealing steps. The optimized staining procedure showed clear and reliable staining patterns and intensities in different species and disease models, and stained a larger number and density of osteocyte lacunae-tubule system networks than the traditional method. Therefore, the optimized new method will provide a more efficient and reliable solution for scientists studying the osteocyte lacunae-tubule system, which will help advance bone biology research and may be more widely used in the diagnosis and treatment of bone-related diseases.

[0010] The first aspect of the present invention provides a method for staining the osteocyte crypt-tubule system, which comprises: (1) preparing (0.5-1) mol / L AgNO3 as solution one, (2) preparing 0.05%-2% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid as solution two; and (3) mixing solution one and solution two at a volume ratio of (4-1):1 and then staining.

[0011] The osteocyte lacuno-canalicular system (LCS) described in the present invention refers to: osteocytes are differentiated and mature osteoblasts, located in small holes in bones called lacunae. A notable feature of osteocytes is that there are a large number of dendrites on the cell surface, called dendrites. These dendrites shuttle through the canaliculi in the bone matrix and connect with other osteocyte dendrites. Therefore, researchers refer to the small holes and canalicular network structure around osteocytes and their dendrites as the osteocyte lacuno-canalicular system (LCS). LCS plays an important role in maintaining bone physiological homeostasis, regulating bone remodeling and osteocyte mechanical conduction processes. There are about 42 billion osteocytes in adult bones, and each osteocyte has about 40-100 dendrites, which extend into the surrounding 20-126 bone canaliculi.

[0012] In a preferred embodiment, the staining method further comprises: staining under a 254 nm UV lamp for 1 hour and staining on a paraffin section.

[0013] In a preferred embodiment, the staining method further comprises: staining for 90 minutes under indoor white light or light-proof conditions; and staining on paraffin sections.

[0014] In a preferred embodiment, the staining method further comprises: terminating the reaction using Milli-Q pure water with a purity of 10-18.2 MΩ·cm, for example, terminating the reaction using Milli-Q pure water with a purity of 11 MΩ·cm, 12 MΩ·cm, 13 MΩ·cm, 14 MΩ·cm, 15 MΩ·cm, 16 MΩ·cm, 17 MΩ·cm, or 18 MΩ·cm.

[0015] In a preferred embodiment, the second solution is selected from the following concentration ranges: 0.05%-1% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid.

[0016] In a preferred embodiment, the type B gelatin has the following concentrations: 0.05% (w / v), 0.1%

[0017] (w / v), 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v), 0.6%

[0018] (w / v), 0.7% (w / v), 0.8% (w / v), 0.9% (w / v) or 1% (w / v) type B gelatin.

[0019] In a preferred embodiment, the formic acid is at the following concentrations: 0.05% (v / v), 0.1% (v / v),

[0020] 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), 0.6% (v / v), 0.7% (v / v), 0.8% (v / v), 0.9% (v / v), 1% (v / v), 1.5% (v / v), 1.8%

[0021] (v / v), 2% (v / v), 2.5% (v / v), 3% (v / v), 3.5% (v / v), 4% (v / v), 4.5% (v / v), or 5% (v / v) formic acid.

[0022] In a preferred embodiment, the second solution is selected from the following concentration ranges: 1%-2% (w / v) type B gelatin and 0.1%-2% (v / v) formic acid.

[0023] In a preferred embodiment, the type B gelatin is at the following concentrations: 0.1% (w / v), 0.2%

[0024] (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v), 0.6% (w / v), 0.7%

[0025] (w / v), 0.8% (w / v), 0.9% (w / v), 1% (w / v), 1.5% (w / v), 1.8% (w / v) or 2% (w / v) of type B gelatin.

[0026] In a preferred embodiment, the formic acid is at the following concentrations: 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), 0.6% (v / v), 0.7% (v / v), 0.8% (v / v), 0.9% (v / v), 1% (v / v), 1.5% (v / v), 1.8% (v / v) or 2% (v / v).

[0027] The second aspect of the present invention provides a kit for specific staining of the osteocyte lacunae-tubule system, the kit comprising a staining solution and a staining stop solution, the staining solution comprising: reagent one: (0.5-1) mol / L AgNO3; and reagent two: a combined solution of 0.05%-2% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid, the volume ratio (v:v) of reagent one and reagent two being (4-1):1; the staining stop solution is 10-18.2 MΩ·cm Milli-Q pure water.

[0028] In a preferred embodiment, the reagent 1 and the reagent 2 are in the form of freeze-dried powders, respectively, and the staining solution is a freeze-dried powder of the reagent 1 and the reagent 2 in the form of freeze-dried powders in a mass ratio (w:w) of (4-1):1.

[0029] In a preferred embodiment, the AgNO3 and type B gelatin-formic acid solutions in the above concentration range are respectively quickly frozen using liquid nitrogen (-196°C) for 30 minutes, and then the fully frozen reagents are vacuum dried at low temperature (-40°C) for 48 hours to obtain the freeze-dried powder form of the reagents one and two.

[0030] In a preferred embodiment, the staining stop solution is used to stop the reaction.

[0031] In a preferred embodiment, the kit further comprises a freeze-dried powder dissolving solution: 10-18.2 MΩ·cm Milli-Q pure water, and 1 ml of freeze-dried powder dissolving solution is added to every 0.1 g of freeze-dried powder to dissolve at room temperature.

[0032] In a preferred embodiment, the second reagent is selected from the following concentration ranges: 0.05%-1% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid.

[0033] In a preferred embodiment, the second reagent is selected from the following concentration ranges: 1%-2% (w / v) type B gelatin and 0.1%-2% (v / v) formic acid.

[0034] The third aspect of the present invention provides a kit for staining the osteocyte lacunae-tubule system, the kit comprising:

[0035] (a) Dye: (0.5-1) mol / L AgNO3 and 0.05%-2% (w / v) B-type gelatin freeze-dried powder dye;

[0036] (b) Lyophilized powder dissolving solution: 0.05%-5% (v / v) formic acid solution;

[0037] and (c) staining stop solution: 10-18.2 MΩ·cm Milli-Q pure water,

[0038] Among them, in the dye, the mass ratio (w:w) of AgNO3 to type B gelatin is (4-1):1.

[0039] In a preferred embodiment, 1 ml (b) of the freeze-dried powder dissolving solution is added to every 0.1 g of the dye to dissolve it at room temperature.

[0040] In a preferred embodiment, the type B gelatin is 0.05%-1% (w / v) or 1%-2% (w / v).

[0041] In a preferred embodiment, the formic acid is 0.05%-5% (v / v) or 0.1%-2% (v / v).

[0042] In a preferred embodiment, the AgNO3 and type B gelatin-formic acid solutions in the above concentration range are respectively quickly frozen using liquid nitrogen (-196°C) for 30 minutes, and then the fully frozen reagents are vacuum dried at low temperature (-40°C) for 48 hours to obtain the lyophilized powder form of the dye.

[0043] In a preferred embodiment, the staining stop solution is used to stop the reaction.

[0044] The fourth aspect of the present invention provides a use of the kit according to the second aspect of the present invention or the kit according to the third aspect of the present invention in staining the osteocyte lacunae-tubule system.

[0045] In a preferred embodiment, staining the osteocyte lacunae-tubule system further comprises a subsequent treatment: staining under a 254 nm UV lamp for 1 hour.

[0046] In a preferred embodiment, staining the osteocyte lacunae-tubule system further comprises subsequent treatment: staining for 90 minutes under room white light or in the dark, and staining on paraffin sections.

[0047] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0048] The reagents and raw materials used in the present invention are commercially available.

[0049] The positive and progressive effects of the present invention are:

[0050] (1) The new method can stain the osteocyte crypt tubules with clear contrast using a (0.5-1) mol / L silver nitrate solution for the first time, while the current method uses a 50% silver nitrate solution (2.943 mol / L), so the amount of silver nitrate used is less, which is more economical.

[0051] (2) The new method determines for the first time the effective range of gelatin and formic acid used, that is, a combination solution of 0.05%-0.5% B-type gelatin and 0.05%-5% formic acid, or a combination solution of 1%-2% B-type gelatin and 0.1%-2% formic acid can obtain clear staining results. The current method uses 2% gelatin and 1% formic acid solution for staining. Therefore, the new method has a more flexible effective concentration range of reagents than the old method, uses less solution, is more economically affordable, reduces pollution to the environment, and is more environmentally friendly.

[0052] (3) The new method determines for the first time the volume ratio of silver nitrate solution to gelatin-formic acid solution. That is, when the volume ratio is 4:1, 2:1 or 1:1, a clear contrast of osteocyte lacunae and tubules can be obtained. The current method only uses a 2:1 ratio. Therefore, the new method has a more flexible range of effective volume ratios than the old method.

[0053] (4) The new method confirmed for the first time that only when gelatin-formic acid solution is prepared using type B gelatin can the staining of osteocyte lacunae with a clear staining background be obtained. When gelatin-formic acid solution is prepared using type A gelatin, a large amount of black silver particles will precipitate on the bone tissue after staining, making it impossible to judge the staining results and the data unusable. However, the current method does not clearly specify the type of gelatin to be used.

[0054] (5) The new method determines for the first time the light conditions and duration for dyeing, that is, 60 minutes under ultraviolet light or 90 minutes under indoor white light or in the dark to obtain effective and clear dyeing, while the current method does not clearly specify the light conditions.

[0055] (6) The new method uses 18.2MΩ·cm Milli-Q pure water to wash after staining to terminate the reaction, and then the contrast-cleaned osteocyte lacunae tubule staining can be obtained. The current method uses 5% sodium thiosulfate solution to terminate the reaction, and then needs to use deionized water to continue washing the slide. In addition, the staining contrast color after the 5% sodium thiosulfate solution terminates the reaction is dim, and the staining effect is not as clear as the new method. The new method uses less solution than the existing method, is more affordable, reduces environmental pollution, and is more environmentally friendly.

[0056] (7) The staining solution in the new method is prepared into a freeze-dried powder dye using vacuum drying technology to form specific dye I and specific dye II. After being dissolved in 18.2MΩ·cm Milli-Q or formic acid solution, the osteocyte crypts and tubules are stained to obtain effective staining with clear contrast. The staining solution in the new method is prepared into a dry powder form, which makes the dye more stable, easier to store and transport, and increases the commercial application potential of the dye reagent.

[0057] In general, the new method is simpler to operate and more affordable than existing methods, and is more environmentally friendly as it uses a lower concentration of silver nitrate solution for staining, a type B gelatin-formic acid solution combination with a more defined concentration range for staining, and 18.2 MΩ·cm Milli-Q pure water for washing to terminate the reaction. It also specifies that effective and clear staining of osteocyte lacunae can be obtained under ultraviolet light for 60 minutes or under indoor white light or in the dark for 90 minutes.

[0058] (8) The new silver nitrate staining method can effectively and clearly stain the lacunae-tubule system of osteocytes in the bones of mammals, amphibians, fish, birds and reptiles. The inventors noticed that compared with other groups of animals, in the silver nitrate staining of the caudal vertebrae of bony fish represented by silver carp, carp and Taihu whitefish, the number of osteocytes was very small, and the number and density of the lacunae-tubule system were very low. This result suggests that the new staining method can provide a powerful staining tool for comparative evolutionary biological studies of osteocytes and lacunae-tubule systems in fish and other groups of animals.

[0059] (9) The new silver nitrate staining method can effectively stain the lacunar canalicular system of osteoblasts in drug-induced disease animal models and transgenic animal models. It is helpful to establish a novel animal model for studying related diseases from a phenotype perspective. For example, the optimized silver nitrate staining method can be used to stain the Prrx1Cre; Zeb1 fx / fx Staining of the cortical bones of gene-specific overexpression mice showed that not only was the density of osteocyte lacunae-tubules significantly reduced, but also a large number of pathological features of cartilage residues appeared in the cortical bones. The staining results obtained using the new method showed for the first time that Prrx1Cre; Zeb1 fx / fx Gene-specific overexpression mice may serve as a new mouse model of osteogenesis imperfecta to further study the expression patterns and related mechanisms of related transcription factors during bone development and osteocyte maturation and differentiation.

[0060] (10) The new method (0.5-1 mol / L silver nitrate) can more effectively stain the lacunae-tubules of osteocytes than the existing method (50% silver nitrate). Statistics show that the LCS density stained with 0.5-1 mol / L silver nitrate is significantly higher than the density of the lacunae-tubules of osteocytes stained with the existing method. It is speculated that during the staining process, the 50% silver nitrate solution in the existing method may not be able to fully present all the lacunae-tubule systems due to its strong reduction reaction, or it is possible that the strong reduction reaction of the high concentration of silver nitrate reagent will damage the lacunae-tubule system. Therefore, there will be deviations when using the existing method to evaluate the number and density of osteocyte lacunae-tubules in different species.

[0061] (11) The optimized new silver nitrate staining method provides an experimental basis for the design and sale of corresponding basic research staining kits or clinical diagnostic kits. Currently, there is no osteocyte lacunae-tubule staining kit on sale in the domestic and foreign markets. The new silver nitrate staining method, specific dye I and specific dye II and their corresponding kits will greatly promote the transformation process of basic research and provide a powerful tool for the diagnosis of specific bone development and bone metabolism diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a flow chart of a novel silver nitrate staining method. This flow chart summarizes the key contents and operation steps of the method involved in this patent, and is intended for use in the study of osteocyte lacunae-tubule morphology staining and quantitative analysis.

[0063] Figure 2 The staining effect of different concentrations of silver nitrate solution on the osteocyte lacunar tubule system when mixed with 2% type B gelatin-1% formic acid solution in different volume ratios. The blue frame range indicates the silver nitrate concentration that can clearly and effectively stain the osteocyte lacunar tubules, as well as the volume ratio of the silver nitrate solution to the gelatin-formic acid solution. Formic acid is formic acid (v / v), and Gelatin is gelatin (w / v). The scale bar is 50μm.

[0064] Figure 3 Comparison of the staining effects of different concentrations of gelatin (0%-5%, w / v) and formic acid (0%-10%, v / v) combined solutions on osteocyte lacunae tubules at a volume ratio of 2:1 with 1 mol / L silver nitrate. The blue frame range is the corresponding gelatin and formic acid concentration combination that can clearly and effectively stain osteocyte lacunae tubules. Formic acid is formic acid (v / v), and Gelatin is gelatin (w / v). The scale bar is 50μm.

[0065] Figure 4The effects of four different conditions on silver nitrate staining of the osteocyte lacunae-tubule system. A: The effects of using 18.2MΩ·cm Milli-Q pure water and 5% sodium thiosulfate aqueous solution to terminate the reaction on silver nitrate staining. B: Comparison of the effects of different types of gelatin (type A gelatin and type B gelatin) on the silver nitrate staining of the osteocyte lacunae-tubule system. The yellow arrow indicates that a large number of black particles are attached to the bone tissue and the slide after staining with type A gelatin. C: The effects of different lighting conditions (UV, or white light, or dark light avoidance) on the silver nitrate staining reaction of the osteocyte lacunae-tubule system. D: The unpaired T test method was used to statistically analyze the silver nitrate staining effects of the osteocyte lacunae-tubules of frozen sections and paraffin sections. E: Statistical analysis of the staining effects of 18.2MΩ·cm Milli-Q pure water and 5% sodium thiosulfate as the termination reaction conditions, n=5. E. The staining effects under three lighting conditions were statistically analyzed using the one-way analysis of variance method, n=4. Scale bar is 50 μm.

[0066] Figure 5 The staining effect of 0.5-1mol / L AgNO3 solution on the crypt-tubule system of the main groups of mammalian osteocytes. The first row from left to right in the figure are the femurs of mouse, rat, and rabbit, the second row are the femurs of bullfrog, wall lizard, and chicken, and the third row are the caudal vertebrae of silver carp, common carp, and Lake Tai white fish. The scale bar is 50μm.

[0067] Figure 6 The lacunae-tubule system of the femoral cortical bone of two different models of mice was stained with 1 mol / L AgNO3 solution. A: The lacunae-tubule system of the femoral cortical bone of the glucocorticoid-induced bone growth retardation mouse model was significantly reduced. B: Transgenic mice that specifically overexpressed the Zeb1 transcription factor in bone mesenchymal stem cells (Prrx1Cre; Zeb1 fx / fx ) of the femoral cortical bone lacunae-tubule system was significantly reduced and sparse. C: Statistical analysis of the femoral osteocyte lacunae-tubule system of mice with glucocorticoid-induced bone growth retardation was performed using the unpaired T test method, n = 5. D. Statistical analysis of the femoral osteocyte lacunae-tubule system of mice with Zeb1 overexpression was performed using the unpaired T test method, n = 4-5. Scale bar is 50 μm.

[0068] Figure 7The two different methods were used to prepare the solution in the new silver nitrate staining method into lyophilized powder dyes and the staining effect of the lyophilized powder dyes on the lacunae tubules of osteocytes. A: Two different operation methods for preparing lyophilized powder dyes and the physical properties of lyophilized powders and the state after dissolution; B: The staining effect of the first lyophilized powder dye on the lacunae tubules of osteocytes; C: The staining effect of the second lyophilized powder dye on the lacunae tubules of osteocytes. The scale bar is 50μm.

[0069] Figure 8 1mol / L silver nitrate can more effectively stain the osteocyte lacunae-tubule system than the classic Ploton silver nitrate staining method (50% silver nitrate). A: The staining results of osteocyte LCS with 1mol / L AgNO3 and 2% gelatin-1% formic acid at different volumes (4:1, 2:1, 1:1), and the third column is the optical density map converted by ImageJ software; B: The staining results of osteocyte LCS with 50% AgNO3 and 2% gelatin-1% formic acid at different volumes (4:1, 2:1, 1:1), and the first column is the optical density map converted by ImageJ software; C: The staining effect of 1mol / L AgNO3 and existing methods on osteocyte LCS was compared using one-way ANOVA, n=4. Scale bar is 20μm. DETAILED DESCRIPTION

[0070] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0071] The following reagents were used in this patent: 10% EDTA decalcification solution (E671001-0500, Sangon Biotech), 10% neutral formalin buffer (311010014, Wexis), sucrose (A100335-0250, Sangon Biotech), OCT Compound (4583, SAKURATissue-Tek), 1M silver nitrate standard solution (P1929026, Bolinda Technology), silver nitrate (C510027-0010, Sangon Biotech), gelatin type A (A609764-0100, Sangon Biotech), gelatin type B (G8061, Solarbio), 88% formic acid (10010118, SinoPharm), sodium thiosulfate pentahydrate (A600484-0500, Sangon Biotech). Biotech), isoflurane (R510-22-10, RWD).

[0072] The following instruments and equipment are used in this patent: biological safety cabinet (Thermo1379 / 1389BiologicalSafety Cabinet with UV light G3675L 31~40W, Thermo), tissue dehydrator (TP1020, Leica), fully automatic paraffin embedding machine (EG1160, Leica), rotary slicer (RM2135, Leica), slice spreader (HistobathHI1210, Leica), frozen slicer (CM1950, Leica), ultrapure water system (Milli-Q Essential, MILLIPORE), upright microscope (ECLIPSE 200, Nikon), vacuum freeze dryer (SCIENTZ-10N / A, Shinji).

[0073] Example 1 Solution preparation and staining process and precautions for the novel osteocyte lacunar tubule silver nitrate staining method

[0074] This patent discloses a new silver nitrate staining method, which makes the staining and characteristic display of the osteocyte lacunar canalicular system in different species and disease models more simple, effective and environmentally friendly. The optimized new method is summarized in detail as follows ( Figure 1 ).

[0075] Solution configuration:

[0076] (1) (0.5-1) mol / L silver nitrate solution: Prepare with 18.2 MΩ·cm Milli-Q pure water and store in dark place at room temperature;

[0077] (2) Gelatin-formic acid solution: First, use 18.2 MΩ·cm Milli-Q pure water to prepare 0.05% to 5% (v / v, such as Figure 3 The concentration of formic acid solution is shown in the blue box line, and then 0.05-2% (w / v, such as Figure 3 Type B gelatin particles (with the concentration shown in the blue box range) were placed in a 0.05% to 5% formic acid solution, fully dissolved in a 37°C water bath for 2 hours, and stored at room temperature after dissolution;

[0078] (3) Staining solution: Mix silver nitrate solution and gelatin-formic acid solution in a ratio of 4:1, 2:1 or 1:1 (v:v). Prepare the solution quickly in a dark place after the sections are dehydrated and before staining. Do not prepare the solution in advance, otherwise the staining effect will be affected.

[0079] Dyeing process:

[0080] (1) Dewaxing: 4 μm paraffin bone sections were dewaxed in xylene three times, 10 min each time;

[0081] (2) Alcohol rehydration: 100%, 90%, 80%, and 70% ethanol, once each, for 5 minutes each time;

[0082] (3) Hydration: Wash twice with 18.2 MΩ·cm Milli-Q pure water for 5 minutes each time. Do not use tap water, PBS, TBS or any solution containing salt for washing.

[0083] (4) Staining: Use absorbent paper to carefully absorb the water around the tissue, place the slide flat on the staining box, prepare the staining solution and drop it on the tissue, and stain under a 254 nm wavelength ultraviolet light in a biosafety cabinet (staining for 60 minutes) or under indoor white light (staining for 90 minutes);

[0084] (5) Termination of staining: After staining, place the slide in 18.2 MΩ·cm Milli-Q pure water and wash on a shaker twice to terminate the reaction, each time for 5 minutes. Do not use tap water, PBS, TBS or any solution containing salt to wash the sections, otherwise precipitation will form on the sections, affecting the data quality.

[0085] (6) Dehydration, transparentization, and sealing: Dehydrate with 70%, 80%, 90%, and 100% once each for 5 minutes, transparentize with xylene three times for 10 minutes each, and seal with neutral dendrites.

[0086] Note:

[0087] (1) Stability of gelatin-formic acid solution The prepared gelatin-formic acid solution can be stored at room temperature for several months. The inventors found that as the storage time increases, the gelatin-formic acid solution may undergo some unknown chemical changes, because when using a 2% gelatin-1% formic acid solution that has been stored for 6 months for staining, the inventors found that it takes longer, such as 2 hours to 3 hours, to obtain the same degree of staining of the osteocyte lacunar tubule system. Therefore, the inventors recommend that the prepared gelatin-formic acid solution be used within 3 months as much as possible, or when it is necessary to use a gelatin-formic acid solution that has been stored for more than 3 months, the staining time can be appropriately extended to obtain a better staining effect.

[0088] (2) Use 18.2MΩ·cm Milli-Q pure water: Tap water, PBS, TBS or any solution containing salt will immediately react with the silver nitrate solution to produce a white flocculent precipitate. Therefore, after dewaxing and rehydration of the sections and after staining, the slides need to be washed with Milli-Q pure water.

[0089] (3) Handling of staining solution: The freshly prepared staining solution (a mixture of silver nitrate and gelatin-formic acid) is initially clear, but turns yellow within 5-10 minutes. The inventors have found that the staining solution that turns yellow is much less effective in staining the osteocyte lacunar canalicular system. Therefore, the inventors recommend dropping the freshly prepared solution onto the slide before the staining solution turns yellow (within 5 minutes, or as soon as possible).

[0090] (4) Slide manipulation During the entire staining process, the slides can be manipulated directly while wearing gloves. There is no need to use non-metallic tweezers or other tools to manipulate the slides.

[0091] Example 2 Optimization and determination of the silver nitrate concentration and volume ratio of the gelatin-formic acid combined solution in the novel method for silver nitrate staining of osteocyte crypt tubules

[0092] This example determines the silver nitrate concentration that can effectively and clearly stain the osteocyte lacunae-tubules: (0.5-1) mol / L silver nitrate and 2% (w / v) gelatin-1% (v / v) formic acid solution can effectively stain the osteocyte lacunae-tubules.

[0093] In the process of establishing the experimental method of silver nitrate staining of the osteocyte lacunae-tubule system, the inventor unexpectedly discovered a staining method that is more concise, effective and economical than the experimental method reported in the classic literature, that is, using 1mol / L silver nitrate solution for staining. On this basis, the inventor systematically studied the effects of different concentrations of AgNO3 (3mol / L, 2mol / L, 1mol / L, 0.5mol / L, 0.25mol / L, 0.1mol / L) and 2% gelatin-1% formic acid solution on the staining of the osteocyte lacunae-tubule system at different volume ratios (4:1, 2:1, 1:1, 1:2, 1:4).

[0094] The results show that ( Figure 2), compared with 50% AgNO3 (2.943mol / L, which can be approximately considered as 3mol / L), 2mol / L and 1mol / L AgNO3 and 2% gelatin-1% formic acid at different volume ratios (4:1, 2:1, 1:1) can produce clearly identifiable staining of the lacunae tubules under UV light for 1 hour. 0.5mol / L AgNO3 can also produce identifiable staining of the lacunae tubules at ratios of 4:1 and 2:1 with 2% gelatin-1% formic acid. Under the condition of staining for 1 hour under UV light, 0.25mol / L and 0.1mol / L AgNO3 concentrations cannot effectively stain the lacunae tubules. In addition, at a specific concentration of AgNO3, the staining depth decreases with the increase in the volume ratio of the 2% gelatin-1% formic acid solution. This suggests that the effective staining of the lacunae tubules is closely related to the concentration of silver nitrate used. When the silver nitrate content is too low, the staining solution cannot effectively stain the lacunae tubules. Based on the effect of lacunae tubule staining and the amount of silver nitrate used, it is believed that when the concentration of 1 mol / L silver nitrate is in a ratio of 2:1 or 1:1 with 2% gelatin-1% formic acid solution, good and relatively complete lacunae tubule staining can be produced when stained under UV light for 1 hour.

[0095] Example 3 Optimization and determination of the concentration range of the gelatin and formic acid combined solution in the novel osteocyte lacunae and tubules silver nitrate staining method

[0096] This example determines the effective concentration range of the gelatin-formic acid combination solution that can effectively and clearly stain the osteocyte lacunae-tubules: when the silver nitrate is (0.5-1) mol / L, the concentration range of 0.05%-1% (w / v) type B gelatin and 0.05%-5% (v / v) FA, or 1%-2% (w / v) type B gelatin and 0.1%-2% (v / v) formic acid solution can effectively stain the osteocyte lacunae-tubule system.

[0097] Based on the conditions in Example 2 that 0.5-1 mol / A AgNO3 is sufficient to fully stain the osteocyte lacunae tubule system, the inventors then compared the effects of different concentration ranges of gelatin (0%-5%, w / v)-formic acid (0%-10%, v / v) solutions on the staining of the osteocyte lacunae tubule system when mixed with AgNO3 solution in a 2:1 or 1:1 volume ratio under UV light for one hour.

[0098] The results show that ( Figure 3), when the gelatin concentration and formic acid concentration are both 0, the silver nitrate solution alone cannot stain the lacunar tubule system of osteocytes. When the gelatin concentration is 0 and the formic acid solution is greater than or equal to 0.05%, although the obvious lacunar tubule system structure can be observed under the microscope after staining, a large amount of black granular precipitates are attached to the entire tissue, rendering the staining data unusable. When the formic acid concentration is 0 and the gelatin solution is greater than or equal to 0.05%, the silver nitrate solution cannot stain the lacunar tubule system of osteocytes. In addition, at a specific formic acid concentration (greater than or equal to 0.05%), the coloring degree of the lacunar tubule system decreases as the gelatin concentration increases. At a specific gelatin concentration (greater than or equal to 0.05%), the coloring degree of the lacunar tubule system first increases and then decreases as the formic acid concentration increases. Experiments show that a combination of 0.05%-0.5% gelatin and 0.05%-5% formic acid, or 1%-2% gelatin and 0.1%-2% formic acid solution can produce clear staining results ( Figure 2 The blue box range corresponds to the combination of gelatin and formic acid concentrations).

[0099] Example 4 The third optimization of the novel osteocyte lacunar tubule silver nitrate staining method

[0100] This embodiment determines the gelatin type, the lighting conditions for staining, the solution used to terminate the staining, and the type of bone section: using type B gelatin, staining under UV for 60 minutes or staining under indoor white light or in the dark for 90 minutes, using 18.2 MΩ·cm Milli-Q pure water to terminate the reaction, and staining on paraffin sections can obtain the best staining effect of the osteocyte lacunae tubule system.

[0101] There are some obvious differences in the silver nitrate staining methods used in different research reports, mainly including the type of gelatin used (type A, type B), lighting conditions (ultraviolet, indoor white light, dark and dark), reaction termination conditions (pure water, 5% sodium thiosulfate), sample processing type (frozen section, paraffin section), etc. Next, the inventors used (0.5-1) mol / LAgNO3 and 2% gelatin-1% formic acid in a 2:1 volume ratio, stained under UV light for 1 hour, and observed the effects of the above four factors on the staining of osteocyte lacunae tubules.

[0102] The results showed that compared with the tissues that were terminated by washing with 5% sodium thiosulfate after staining, the tissues that were terminated by washing with 18.2 MΩ·cm Milli-Q pure water showed a clearer and brighter coloration, but there was no visible difference in the density of the lacunar tubule system stained by the two methods ( Figure 4 A and Figure 4E). When different types of gelatin were used to prepare the staining solution, the results showed that a large amount of black particles would be attached to the tissue after type A gelatin staining, while the staining background of both brands of type B gelatin was clear without any black particles ( Figure 4 B). Therefore, it is speculated that the black particles in the silver nitrate staining images in some bone cell research literature may be caused by the use of inappropriate gelatin types.

[0103] When different lighting conditions were used for staining for 1 hour at the same time, the results showed that the staining of the crypt tubule system under UV conditions (254nm, 31-40W) and indoor white light was clearer than that under dark conditions ( Figure 4 Although no difference in the staining density of the pit tubule system was observed under the three lighting conditions for 1 hour, the inventors found that the depth of UV staining of the pit tubule system was higher under indoor white light and dark conditions ( Figure 4 F). When stained for 90 minutes, the depth of staining of the crypt tubule system under indoor white light and dark conditions can reach the depth of staining under UV irradiation for 1 hour. In addition, both frozen sections and paraffin sections can show clear contrast staining, but the crypt tubule system of paraffin sections is more orderly, while the crypt tubule system of frozen sections looks scattered and disordered. It is speculated that this may be due to the well-known fact that frozen sections are not as effective in preserving the overall morphology of tissues as paraffin sections ( Figure 4 D). Therefore, the data of this embodiment show that type B gelatin, staining under UV or white light, using 18.2MΩ·cm Milli-Q pure water to terminate the reaction, and staining on paraffin sections are the conditions that guarantee the best staining effect of the osteocyte lacunar tubule system.

[0104] Example 5 The optimized new silver nitrate staining method can clearly stain the lacunar canalicular system of osteocytes of different species

[0105] In order to verify the universality of the optimized staining method, the inventors performed silver nitrate staining of the osteocyte lacunar tubule system on bone samples of different species. The staining solution was prepared using a 2:1 or 1:1 volume ratio of 0.5-1mol / LAgNO3 and 2% B-type gelatin-1% formic acid solution, and the reaction was carried out under UV irradiation for 1 hour. After the reaction, 18.2MΩ·cm Milli-Q pure water was used for washing to terminate the reaction.

[0106] The staining results showed that ( Figure 5), the new method stained clear osteocyte lacunae-tubule systems in major vertebrate groups such as mammals (mice, rats, rabbits), amphibians (bullfrogs), fish (silver carp, carp, Taihu whitefish), birds (chickens) and reptiles (geckos). At the same time, the staining results showed that compared with other vertebrates, the number of osteocytes in the spine of bony fish represented by silver carp, carp and Taihu whitefish was very small, and the number and density of lacunae-tubule systems were very low. This shows that bony fish generally lack differentiated and mature osteocyte synaptic structures and lacunae-tubule systems, and this feature may be related to the adaptation of fish to the buoyant aquatic living environment.

[0107] Example 6 The optimized new silver nitrate staining method can effectively stain the osteocyte lacunar canalicular system in the glucocorticoid-induced bone growth arrest mouse model and the transgenic mouse model

[0108] The bone growth retardation model was established by intraperitoneal injection of dexamethasone (2 mg / kg / day) in 4-week-old male mice. The model showed pathological characteristics of slow growth of body weight, femur and tibia. fx / fx The transgenic mouse model (Prrx1Cre; Zeb1) that specifically overexpresses Zeb1 in bone mesenchymal stem cells was obtained by mating mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). fx / fx ), which exhibits pathological features of osteogenesis imperfecta.

[0109] To evaluate the potential application value of the optimized staining method in pathological models, silver nitrate staining analysis was performed on bone samples from two different mouse animal models: a glucocorticoid-induced bone growth arrest model mouse and a transgenic mouse that overexpressed the Zeb1 transcription factor specifically in bone mesenchymal stem cells (Prrx1Cre; Zeb1 fx / fx ). The dye solution was prepared by mixing 0.5-1mol / LAgNO3 and 2% B-type gelatin-1% formic acid solution in a volume ratio of 2:1 or 1:1, and the reaction was irradiated with UV for 1 hour. After the reaction, only Milli-Q pure water was used to wash to terminate the reaction. The staining showed that the optimized staining method can effectively stain the osteocyte crypt tubule system of the two model mice ( Figure 6 A and Figure 6 B) Compared with control mice, dexamethasone mice and Prrx1Cre;Zeb1 fx / fx The density of the lacunar tubule system in mice was significantly reduced ( Figure 6 C and Figure 6 D). In addition, Prrx1Cre; Zeb1 fx / fxThe staining results of gene-specific overexpression mice suggest that overexpression of Zeb1 may hinder the normal differentiation of mesenchymal stem cells into osteoblasts and osteocytes, leading to an imbalance in the differentiation of chondrocytes and osteoblasts, resulting in the pathological characteristics of cartilage residues in the cortical bones of adult mice. These results further indicate that the optimized staining method can effectively distinguish changes in the lacunar canalicular system in common animal pathological models.

[0110] Example 7 Specific dyes I and II in the form of freeze-dried powder can effectively stain the lacunar canalicular system of osteocytes

[0111] Dyes in dry powder form have become the most commonly used commodity form in the industrial production and sales of modern biological reagents due to their advantages such as easy storage and transportation, greater stability, long shelf life, and ease of configuration and concentration control according to experimental needs. In order to explore the commercial application potential of the new staining method, the inventors further explored the possibility of preparing the staining solution into a freeze-dried powder for staining.

[0112] The inventors prepared the dye solution into a freeze-dried powder form by two methods ( Figure 7 A): (1) Specific dye I, a freeze-dried powder dye of a combined solution of (0.5-1) mol / L AgNO3 and 0.05%-2% (w / v) type B gelatin-0.05%-5% (v / v) formic acid; (2) Specific dye II, a freeze-dried powder dye of (0.5-1) mol / L AgNO3 and 0.05%-2% (w / v) type B gelatin. The solutions mentioned in the two methods are mixed evenly, quickly frozen with liquid nitrogen (-196°C) for 30 minutes, and then the fully frozen staining reagent is vacuum dried at low temperature (-40°C) for 48 hours, so that specific dye I and specific dye II can be obtained. Before staining, weigh 0.1g of specific dye I, dissolve it in 1ml of 18.2MΩ·cm Milli-Q pure water, and stain it at room temperature and indoor white light for 2 hours to complete the staining. When using specific dye II, weigh 0.1g of specific dye II, dissolve it in 1ml of 0.05%-5% (v / v) formic acid solution, and then stain it at room temperature under indoor white light for 2 hours to complete the staining. The staining results show that specific dye I and specific dye II can effectively and clearly stain the osteocyte crypt-tubules ( Figure 7 B and Figure 7 C). The staining effect of the two dry powder forms is comparable to that of the dye solution prepared directly using silver nitrate and gelatin-formic acid.

[0113] Therefore, the solution in the new staining method is prepared into specific dye I and specific dye II in the form of freeze-dried powder, which are then dissolved before use. This method makes the dye properties more stable and easier to store and transport, increasing the commercial application potential of the dye reagent.

[0114] Comparative Example 1 The optimized new silver nitrate staining method (0.5-1 mol / L silver nitrate) can more effectively stain the osteocyte lacunar canalicular system than the existing silver nitrate staining method (50% silver nitrate)

[0115] The differences in staining the osteocyte lacunar tubule system between the optimized new method (0.5-1 mol / LAgNO3) and the existing staining method (50% AgNO3) were further compared. Different concentrations of silver nitrate solution (1 mol / LAgNO3 and 50% AgNO3) were mixed with 2% type B gelatin-1% formic acid solution in different volumes (4:1, 2:1, 1:1), and UV irradiated for 1 hour. After the reaction, only Milli-Q pure water was used for washing to terminate the reaction. Statistical analysis showed that ( Figure 8 C), when the ratio with 2% gelatin-1% formic acid solution is 4:1 or 2:1, the density of the osteocyte lacunae tubule system stained with 0.5-1mol / LAgNO3 is significantly higher than that of the existing silver nitrate staining method (50% AgNO3). The osteocyte lacunae tubule system stained with 0.5-1mol / LAgNO3 is more complete and clear ( Figure 8 A), while the osteocyte lacunar tubule system stained by Ploton silver nitrate staining (50% AgNO3) is intermittent, irregular, and has a blurred contrast ( Figure 8 B).

[0116] In addition, statistical analysis showed that when the existing staining solution and its ratio were used, that is, when the volume ratio of 50% AgNO3 to 2% gelatin-1% formic acid solution was 2:1, the density of the stained osteocyte lacunae tubule system was significantly lower than that of the stained osteocyte lacunae tubule system when the volume ratio of 0.5-1mol / L AgNO3 to 2% gelatin-1% formic acid solution was 4:1, 2:1, and 1:1 ( Figure 8C). This result suggests that high-concentration silver nitrate staining solution may cause certain damage to the lacunar tubule system during the staining process, or that the high-concentration silver nitrate is strongly reduced in the reaction, resulting in a too fast reaction rate, and all the lacunar tubule systems cannot be fully stained. The inventor speculates that this may be a major reason why the 0.5-1mol / LAgNO3 used in the new method can more effectively stain the lacunar tubule system of osteocytes than the existing silver nitrate staining method (50% AgNO3). Therefore, this is the first time that experiments have shown that the existing staining method (50% AgNO3) has a significant destructive effect on the lacunar tubule system of osteocytes during staining, and thus this method will lead to an underestimation of specific values ​​when quantitatively analyzing and comparing the lacunar tubule system of a specific species. The new method of the present invention can not only stably and clearly stain the lacunar tubule system, but also effectively avoid the destructive effects of traditional methods, and thus can more accurately quantitatively analyze and compare the lacunar tubule system of a specific species.

Claims

1. A method for staining the osteocyte lacunae-tubule system, comprising: (1) preparing (0.5-1) mol / L AgNO3 as solution one, (2) preparing 0.05%-2% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid as solution two; (3) mixing solution one and solution two at a volume ratio of (4-1):1 and then dyeing.

2. The method according to claim 1, characterized in that The staining method further comprises: staining under a 254 nm UV lamp for 1 hour, or staining under indoor white light or light-proof conditions for 90 minutes; and staining on paraffin sections.

3. The method according to claim 1 or 2, characterized in that The staining method further comprises: using Milli-Q pure water with a purity of 10-18.2 MΩ·cm to terminate the reaction.

4. The method according to claim 3, characterized in that The second solution is selected from one of the following concentration ranges: 0.05%-1% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid; Alternatively, 1%-2% (w / v) type B gelatin and 0.1%-2% (v / v) formic acid.

5. A kit for specific staining of the osteocyte lacunae-tubule system, characterized in that: The kit includes a staining solution and a staining stop solution, wherein the staining solution includes: reagent one: (0.5-1) mol / L AgNO3; and reagent two: a combined solution of 0.05%-2% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid, wherein the volume ratio (v:v) of the reagent one to the reagent two is (4-1):1; and the staining stop solution is 10-18.2 MΩ·cm Milli-Q pure water.

6. The kit according to claim 5, characterized in that The reagent 1 and the reagent 2 are in the form of freeze-dried powders, respectively. The staining solution is freeze-dried powders of the reagent 1 and the reagent 2 in the form of freeze-dried powders in a mass ratio (w:w) of (4-1):

1.

7. The kit according to claim 6, characterized in that The kit also includes a freeze-dried powder dissolving solution: 10-18.2 MΩ·cm Milli-Q pure water, 1 ml of the freeze-dried powder dissolving solution is added to every 0.1 g of freeze-dried powder to dissolve at room temperature.

8. The kit according to any one of claims 5 to 7, characterized in that Reagent 2 in the kit is selected from one of the following concentration ranges: 0.05%-1% (w / v) type B gelatin and 0.05%-5% (v / v) formic acid; Alternatively, 1%-2% (w / v) type B gelatin and 0.1%-2% (v / v) formic acid.

9. A kit for staining the osteocyte lacunae-tubule system, characterized in that: The kit comprises: (a) Dye: (0.5-1) mol / L AgNO3 and 0.05%-2% (w / v) B-type gelatin freeze-dried powder dye; (b) Lyophilized powder dissolving solution: 0.05%-5% (v / v) formic acid solution; and (c) staining stop solution: 10-18.2 MΩ·cm Milli-Q pure water, Among them, in the dye, the mass ratio (w:w) of AgNO3 to type B gelatin is (4-1):

1.

10. The kit according to claim 9, characterized in that Add 1 ml (b) of freeze-dried powder dissolving solution to every 0.1 g of dye and dissolve at room temperature.

11. The kit according to claim 9 or 10, characterized in that The type B gelatin is 0.05%-1% (w / v) or 1%-2% (w / v); and / or the formic acid is 0.05%-5% (v / v) or 0.1%-2% (v / v).

12. Use of the kit according to any one of claims 5 to 8 or the kit according to any one of claims 9 to 11 for staining the osteocyte lacunae-tubule system.

13. The use according to claim 12, characterized in that Staining of the osteocyte lacunae-tubule system also includes subsequent staining under 254 nm UV light for 1 hour; alternatively, staining under room white light or in the dark for 90 minutes and staining on paraffin sections.

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