A quantitatively optimized method and kit for preparing transparent fish skeleton specimens stained with Alcian Blue and Alizarin Red.

Abstract

This invention provides a standardized preparation method and kit for double-stained transparent specimens of fish skeletons. Orthogonal experiments were designed for fish of different skeleton types (cartilaginous / boneless) and body lengths (small 5-7cm, medium 7-15cm, large 15-25cm), using factors such as KOH concentration and treatment time. Staining effect, transparency, and tissue integrity were used as indicators. Statistical analysis was used to establish an optimal parameter matrix database covering fixation, transparentization, and staining steps. During preparation, the database can be queried based on fish species and body length to obtain differentiated treatment parameters, achieving standardized operation. This invention also provides a kit containing staining stock solution, basic reagents, and a parameter query unit. This invention transforms double-stained skeleton preparation from experience-dependent to data-driven, significantly improving repeatability and success rate, and is suitable for scientific research, teaching, and demonstration.

Description

Technical Field

[0001] This invention belongs to the field of biological specimen preparation technology and histochemical staining, specifically involving a method for clearing and differential staining of fish skeletal specimens. In particular, it is a standardized preparation method and matching kit for Alcian Blue-Alizarin Red S double-stained clear specimens of cartilaginous and bony fish of different sizes (small, medium and large fish) by establishing a database of key step parameter matrices through a multi-factor orthogonal optimization model. Background Technology

[0002] Alixin Blue-Alizarin Red double staining is a classic histological technique for distinguishing and displaying cartilage (stained blue) and bone (stained red) in animals. Its application in the preparation of transparent whole fish specimens allows for clear visualization of the three-dimensional structure and developmental state of the skeletal system while preserving the tissues in situ, making it invaluable in fish morphology, taxonomy, developmental biology, and educational demonstrations.

[0003] However, traditional methods for preparing double-stained transparent specimens of fish skeletons have the following technical drawbacks: (1) Parameter selection relies on experience and lacks systematic data support. In existing methods, key steps such as fixation, clearing, staining, and destaining, along with processing times and reagent concentrations, are largely based on operator experience or generic formulations. They lack systematic quantitative studies addressing the physiological differences between different fish species (especially the fundamental differences between cartilaginous and bony fish in terms of skeletal matrix composition and soft tissue density) and individual body size. This results in poor reproducibility and generalizability, with significant variations in effectiveness between different operators or different batches of specimens. (2) The interaction of key factors was not revealed. Complex interactions exist among several key factors affecting staining outcomes (such as KOH concentration, treatment time, staining time, and destaining time). Traditional methods, employing single-factor experiments or fixed parameter combinations, fail to reveal the interactive effects between factors and struggle to obtain the truly optimal process combination. For example, high-concentration KOH treatment for a short time and low-concentration KOH treatment for a long time may achieve similar transparency results, but their impact on tissue integrity is drastically different. (3) No differentiated parameter system was established for fish type and body size. The skeletal composition of cartilaginous and bony fish differs significantly: cartilaginous fish skeletons are predominantly cartilaginous, while bony fish skeletons are predominantly bony. This leads to fundamentally different dynamic behaviors in the clearing and staining processes between the two types of fish. Furthermore, individuals of different sizes exhibit significant differences in tissue thickness and permeability; using the same treatment parameters inevitably results in overtreatment of smaller individuals and undertreatment of larger individuals. No systematic and differentiated parameter systems for these two types of variables have been reported in existing technologies. (4) Lack of standardized operating instructions products The entire process involves the repeated preparation and replacement of various reagents, is lengthy, and can take several weeks in total. There are currently no standardized products on the market that integrate optimized parameters and pre-prepared key reagents for this method, which hinders the efficient and stable application of this technology in scientific research and teaching.

[0004] Therefore, there is an urgent technical need to develop a method for preparing double-stained transparent skeletal specimens based on a multi-factor quantitative optimization model with well-defined parameters, standardized operation, and applicable to different fish types and body sizes, as well as its matching reagent kit.

[0005] The purpose of this invention is to provide a quantitatively optimized method for preparing transparent fish skeleton specimens stained with Alcian Blue and Alizarin Red, in order to solve the problems in the prior art, such as reliance on experience for parameter selection, neglect of the interaction of key factors, lack of differentiated parameter systems for cartilaginous and bony fish and different body sizes, and lack of standardized operation guidance products. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the objective of this invention is: This invention provides a quantitative, standardized, and reproducible method for preparing fish skeletons double-stained clear specimens using Alizonium Blue and Alizarin Red. Through systematic comparative experiments, this method establishes the optimal reagent concentration and processing time parameters for each key step (fixation, pre-clearing, staining, destaining, and re-clearing) for both cartilaginous and bony fish, as well as different body sizes (small, medium, and large).

[0007] Based on the above optimized parameters, a dedicated integrated reagent kit was developed. This kit includes pre-prepared core staining solutions and calculated quantities of basic reagents, along with detailed operating procedures and parameter lookup tables, aiming to achieve "ready-to-use" operation, significantly simplifying the process, shortening the total time, and lowering the technical threshold.

[0008] Ultimately, a simple, efficient, stable, and convenient method for preparing double-stained transparent specimens of fish skeletons was achieved, ensuring the acquisition of high-quality specimens with high staining contrast, intact skeletal structure, and clear transparent background, thus meeting the diverse needs of scientific research, teaching, and exhibition.

[0009] The technical solution adopted in this invention is to transform the traditional experience-driven fish skeleton staining process into a quantitative and standardized system based on systematic experimental data, and to develop a corresponding integrated reagent kit. Its innovation is specifically reflected in: (I) Construction of a multi-factor optimization parameter matrix database This invention first provides a method for constructing a multi-factor optimization parameter matrix database, which transforms fish specimen preparation from experience-driven to data-driven, specifically including the following steps: Determination of S100 categorical variables Fish are classified into two types of skeletons: cartilaginous fish and bony fish. For each type of skeleton, fish are divided into at least three grades based on body length, which include: small fish 5-7cm, medium fish 7-15cm, and large fish 15-25cm. S200 Multifactor Orthogonal Experimental Design For each grade, key process parameters affecting staining results were selected as experimental factors. Each factor was configured with three levels, and an orthogonal array was used to design the experimental scheme. These key process parameters included: KOH concentration, pre-clearing treatment time, cartilage staining time, bone staining time, and decolorization time, etc. (orthogonal array type and factor level table, such as...) Figure 6-9 (as shown) Determination of S300 evaluation indicators The staining effect, muscle transparency, and tissue structure integrity were used as evaluation indicators for process optimization. in: Staining: A quantitative amount of stained bone was taken and thoroughly destained, and the absorbance of the destained solution was compared with that of the unstained bone destained solution. Muscle transparency: By taking muscle samples from fish, measuring the thickness, and using a parallel light source and a light intensity meter, the results were substituted into a formula. Obtain the light absorption coefficient of muscle tissue, where T: Transmittance, obtained through the formula (T= (×100%) R: Reflectance, expressed by the formula (R= )get α: Light absorption coefficient of the material x: Sample thickness; Tissue structure integrity rate: refers to the proportion of bone structures that retain their original morphological integrity after clearing and staining. It is used to assess the degree of damage to bone tissue caused by the manufacturing process. Observation and Counting Methods: The processed samples were placed under a stereomicroscope (magnification 10×-40×) to systematically observe the main skeletal structures of the fish. The following representative skeletal structures were selected as counting objects: each bone of the skull, the vertebral column (each vertebra), each rib, spinal spines, vascular spines, and each fin ray; Damage assessment criteria: Under a microscope, any of the following conditions constitute an "incomplete skeletal structure": Fracture: The bone shows obvious transverse or longitudinal cracks, and the continuous structure is interrupted; Dislocation: The bone detaches from its original position and loses its connection with the surrounding bones; Dissolution: Obvious corrosion, defects, or partial disappearance of the bone edges, and the surface is no longer smooth; Calculation formula: Organizational structure integrity rate (%) =(1- ) × 100% At least 50 clearly identifiable skeletal structures were counted in each sample, and the counts were made independently by two observers, with the average value taken as the final result. It should be noted that the methods described above for measuring staining effect and transparency are mainly used in the multi-factor orthogonal experimental stage when constructing the parameter matrix database in this invention. The purpose is to objectively and quantitatively screen out the optimal process parameters. Once the database is constructed, the operator does not need to repeat the above quantitative measurements when preparing standardized specimens according to the embodiments of this invention. Whether the final specimen achieves the visual effect of "cartilage appearing bright blue, bone appearing clear red, and muscle tissue being transparent" is the intuitive basis for verifying whether the process parameters meet the standards. Implementation and Data Acquisition of S400 Orthogonal Experiment The experiment was conducted according to the orthogonal experimental design table, and samples were obtained under each experimental combination. The specific values ​​of the three evaluation indicators mentioned above were then measured. S500 Statistical Analysis and Determination of Optimal Parameters Range and variance analyses were performed on the experimental results to determine the significance order of the influence of each factor on the evaluation indicators, as well as the optimal combination of levels for each factor. Taking into account the three evaluation indicators, the optimal parameter combination for each level was determined, forming the parameter matrix database. Structure of the S600 parameter matrix database The parameter matrix database is indexed by fish type (cartilaginous fish / boneless fish) and body length grade (small / medium / large), storing the optimal processing parameters for each key step, including but not limited to: Total time for alcohol gradient fixation; KOH pre-transparency concentration and treatment time; Cartilage staining time; Decolorization time and fluid change frequency for cartilage; Re-transparency concentration and processing time; Staining time for bone hardness; Decolorization time and solution change frequency for bone hardening; Time required for muscle recoloring and bleaching; Total time for gradient glycerol to become clear.

[0010] (II) Standardized preparation method based on parameter matrix database Based on the parameter matrix database constructed above, this invention provides a standardized method for preparing double-stained transparent specimens of fish skeletons, comprising the following steps, wherein the specific processing parameters for each step are selected and determined from the parameter matrix database according to the skeleton type and body length grading of the target fish: S1 Sample Preprocessing Remove the skin and internal organs of the fish, keeping the skeletal structure intact, and gently rinse the body cavity with distilled water; S2 alcohol gradient fixation The samples were sequentially fixed using alcohol solutions of 50%, 75%, and 95% concentrations. S3 KOH pre-transparent The sample was treated with KOH solution to make the muscle tissue appear translucent. S4 cartilage staining Alcian blue staining solution was used to stain the cartilage tissue; S5 Cartilage Decolorization Decolorization was performed using a 95% alcohol solution; S6 Transparent The sample was then cleared again using KOH solution; S7 bone staining Alizarin red staining solution was used to stain the bone tissue; S8 Hard Bone Decolorization Decolorization was performed using distilled water; S9 Muscle Recoloring and Bleaching The sample was decolorized and bleached using a 3% hydrogen peroxide solution; S10 gradient glycerol clear The samples were treated sequentially with gradient clearing solutions of 25%, 50%, and 75% glycerol; S11 Save The specimen was preserved in pure glycerol containing thymol.

[0011] (III) Reagent Kit To standardize the operation of the above method, this invention also provides a kit for preparing double-stained transparent specimens of fish skeletons, comprising: (1) Staining reagent unit Alixin Blue staining stock solution (100× concentrate): Each 5 mL stock solution contains 50 mg of Alixin Blue and 5 mL of ethanol; Alizarin Red staining stock solution (100× concentrate): Each 5 mL of stock solution contains 0.05 mg of alizarin red; (2) Basic Reagent Unit KOH solid (analytical grade): used to prepare the transparent working solution; Ethanol (volume fraction ≥95%): used for fixation and decolorization; Glycerin (volume fraction ≥99.5%): used for final clarification and preservation; Hydrogen peroxide (30% concentration): used for decolorization and bleaching; Glacial acetic acid (concentration ≥97%): used for preparing staining solutions and adjusting pH; (3) Standardization guidance unit The instruction manual includes a lookup table for the parameter matrix database and an operation manual. The lookup table is indexed by fish type (cartilaginous / boneless) and body length grade (small / medium / large), listing the optimal processing parameters for each key step for direct use by the operator.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: Standardization and high success rate: By providing precise quantitative parameters, the reliance on the operator's personal experience in traditional methods is completely eliminated, greatly improving the reproducibility and success rate of experiments, and ensuring that high-quality specimens with clear staining contrast, transparent background and complete structure can be obtained by different operators and different laboratories.

[0013] High efficiency and low cost: The optimized parameter system ensures the best staining effect while scientifically shortening unnecessary processing time (for example, clarifying the optimal fixation and decolorization endpoints for various fish species), resulting in a significant reduction in the overall preparation cycle. The integrated kit avoids the tedious steps of researchers searching for and preparing multiple reagents themselves, saving time and material management costs.

[0014] Easy to operate and promote: The reagent kit integrates complex multi-step processes into a clear standardized process, which greatly reduces the technical threshold and makes the technology easier to promote and use in teaching, popular science and standardized scientific research.

[0015] This invention reveals the interaction of factors and provides a theoretical basis: Through range analysis and variance analysis of orthogonal experiments, it reveals for the first time the significance order and interaction of the effects of key factors on staining results. For example, for the KOH pre-clearing step in bony fish, the significance order of factors is: treatment time > KOH concentration > fish size, and there is a significant interaction between treatment time and KOH concentration (P < 0.05). This finding provides a theoretical basis for further optimization of the process.

[0016] Wide applicability and reliability: This invention is the first to systematically cover both cartilaginous and bony fish groups and their complete size range, providing targeted solutions. All parameters are derived from comparative experimental data of the system, ensuring the scientific validity of the solution and the stability and reliability of the results. Attached Figure Description

[0017] Figure 1 : Overall process flow diagram of the fish skeleton transparency staining method of the present invention; Figure 2 Example of a flowchart illustrating specific operating parameters for small cartilaginous fish (5-7cm); Figure 3 Example of a flowchart illustrating specific operating parameters applicable to medium-sized bony fish (7-15cm); Figure 4 Example of a flowchart illustrating specific operating parameters for large cartilaginous fish (15-25cm); Figure 5 The core of this invention is a complete parameter matrix database table. This table is indexed by fish species (cartilaginous fish / boneless fish) and body size (small / medium / large). It lists the optimized parameter set for reagent concentration, treatment time and liquid change frequency for each step, which is the direct basis for implementing standardized operations. Figure 6 Table of factor levels in orthogonal experiment of KOH pre-transparency treatment for small and medium-sized bony fish; Figure 7 Table of factor levels in orthogonal experiment of KOH pre-clearing treatment for large bony fish; Figure 8 Table of KOH pre-transparency orthogonal experiment results and range analysis for small-sized bony fish; Figure 9 Table of KOH pre-transparency orthogonal experimental results and range analysis for large bony fish; Figure 10 Table of factor levels in orthogonal experiment for KOH pre-clearing treatment of small and medium-sized cartilaginous fish; Figure 11 Table of factor levels in orthogonal experiment of KOH pre-clearing treatment for large cartilaginous fish; Figure 12Table of KOH pre-transparency orthogonal experimental results and range analysis for small and medium-sized cartilaginous fish; Figure 13 Table of KOH pre-transparency orthogonal experimental results and range analysis for large cartilaginous fish; Figure 14 : Graph showing the change in cartilage staining effect over time for different body sizes of bony fish; Figure 15 Graph showing the change in cartilage staining effect over time for different body sizes of cartilaginous fish; Figure 16 Graph showing the change in cartilage decolorization effect over time for different body sizes of bony fish; Figure 17 Graph showing the change in cartilage decolorization effect over time for different body sizes of cartilaginous fish; Figure 18 Graph showing the change in the transparency of small bony fish with different concentrations of KOH over time; Figure 19 Graph showing the change in the re-transparency effect of different concentrations of KOH in bony fish over treatment time; Figure 20 Graph showing the change in the transparency of large bony fish with different concentrations of KOH over time; Figure 21 Graph showing the change in the transparency of small cartilaginous fish with different concentrations of KOH over time; Figure 22 Graph showing the change in the re-transparency effect of different concentrations of KOH in cartilaginous fish over treatment time; Figure 23 Graph showing the change in the transparency of large cartilaginous fish with different concentrations of KOH over time; Figure 24 : Graph showing the change in bone staining effect over time for different body sizes of bony fish; Figure 25 : Graph showing the change in bone staining effect over time for different body sizes of cartilaginous fish; Figure 26 Graph showing the change in decolorization effect of bony fish of different body sizes over time; Figure 27 Graph showing the change in decolorization effect of cartilaginous fish of different body sizes over time. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments. In this invention, 'small fish' refers to individuals with a body length of 5-7 cm; 'medium fish' refers to individuals with a body length of 7-15 cm; and 'large fish' refers to individuals with a body length of 15-25 cm. The body length is the straight-line length from the snout to the end of the caudal fin. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the scope of protection of the invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] Example 1: Preparation of a double-stained transparent specimen of the skeleton of a cartilaginous fish (small, 5-7 cm in length) The embodiments illustrate in detail the optimized operating procedures for juvenile cartilaginous fish. See [link to documentation]. Figure 2 The method includes the following steps: 1. Sample preprocessing Take one fresh or preserved sturgeon (cartilaginous fish), about 7cm in length. Carefully remove the skin, gills, and internal organs using dissecting instruments, taking care to avoid damaging the spine, fins, skull, and other skeletal structures. Gently rinse the body cavity with distilled water; 2. Alcohol gradient fixation (1) Preparation of fixative: Take 21 mL of 95% alcohol from the kit, add 19 mL of distilled water, mix well and prepare 40 mL of 50% alcohol solution; (2) Gradient fixation: The treated fish samples were completely immersed in the above 50% alcohol solution and fixed at room temperature; (3) Change the fixative: According to the experimental data, after 2 days of fixation, change the fixative to 75% alcohol (prepared with 95% alcohol from the kit); after 4 days of fixation, change it to 95% alcohol again; (4) Fixation endpoint: The total fixation time is 8 days. After fixation, the samples are removed and placed in a 40℃ oven for gentle drying for 5 minutes to remove excess alcohol from the surface; 3. KOH pre-transparency (1) Preparation of clear solution: Weigh 1 g of KOH solid from the kit and dissolve it in 100 mL of distilled water to prepare a 1% KOH solution; (2) Transparency treatment: The fixed and dried fish samples are completely immersed in the above KOH solution; (3) Processing time: According to the optimized parameters, process for 2.5 hours at room temperature. Gently shake the fish during this time, and stop processing when the fish muscles become translucent. (4) Washing: Transfer the sample to a large amount of distilled water, soak and change the water several times to thoroughly wash away any residual KOH; 4. Cartilage staining (alicin blue staining) (1) Preparation of dye solution: Measure 0.5 mL of 100× Alcian Blue stock solution from the kit and add it to 40 mL of 95% ethanol and 9.5 mL of glacial acetic acid solution. Mix well to prepare 50 mL of working solution. Adjust the pH of the dye solution to between 4.2 and 5.8 with KOH solution. (2) Staining treatment: Immerse the cleaned sample in the staining solution; (3) Processing time: According to the optimized parameters, staining time is 16 hours; 5. Cartilage decolorization (1) Decolorization treatment: Take out the stained sample and immerse it in 40 mL of 95% alcohol for decolorization; (2) Change the decolorizing solution: Change the decolorizing solution every 6 hours until the alcohol solution is basically colorless; (3) Decolorization endpoint: According to the optimized parameters, the total decolorization time is approximately 24 hours; 6. Re-transparency (KOH becomes transparent again) (1) Preparation of clear solution: Weigh 1 g of KOH solid from the kit and dissolve it in 100 mL of distilled water to prepare a 1% KOH solution; (2) Re-transparency treatment: Immerse the decolorized sample in the above solution; (3) Processing time: Based on the optimized parameters, the processing time is 3 hours. After completion, rinse briefly with distilled water; 7. Staining of bone (Alizarin Red staining) (1) Preparation of dye solution: Measure 0.5 mL of 100×Alizarin Red stock solution from the kit, add it to 49.5 mL of distilled water, add 0.5 g KOH, mix well to prepare 50 mL of working solution; (2) Staining treatment: Immerse the cleared sample in the staining solution; (3) Processing time: According to the optimized parameters, staining time is 20 hours; 8. Bone decolorization (1) Decolorization treatment: Take out the stained sample and immerse it in 100 mL of distilled water for decolorization; (2) Change the decolorizing solution: Change the decolorizing solution every 8 hours; (3) Decolorization endpoint: According to the optimized parameters, the total decolorization time is approximately 48 hours; 9. Muscle recoloring and bleaching (1) Preparation of bleaching solution: Measure 5 mL of 30% hydrogen peroxide solution from the kit and add 45 mL of distilled water to prepare 50 mL of 3% hydrogen peroxide solution; (2) Bleaching treatment: Immerse the sample in the above solution and place it under soft light; (3) Treatment endpoint: Soak until the muscle tissue is nearly colorless and the blue cartilage and red bone are clearly visible. After completion, rinse thoroughly with distilled water; 10. Gradient Glycerin Clearing and Storage (1) Gradient solution preparation: - 25% glycerol: Measure 12.5 mL of glycerol from the kit, add 37.5 mL of 0.5% KOH solution (prepared from the KOH in the kit), and mix well; - 50% glycerol: Measure 25 mL of glycerol, add 25 mL of 0.5% KOH solution, and mix well; - 75% glycerol: Measure 37.5 mL of glycerol, add 12.5 mL of 0.5% KOH solution, and mix well; - Pure glycerol preservation solution: Add thymol to pure glycerol until saturated (about 1-5 mg / mL) to prepare a long-term preservation solution; (2) Gradient treatment: Immerse the sample in 25% glycerol, 50% glycerol and 75% glycerol for 24 hours each. Finally, transfer to 75% glycerol, changing the solution once a day until the sample is completely transparent (approximately 4 days). (3) Sealing: Transfer the completely transparent specimen to pure glycerol preservation solution containing thymol, and seal it in a specimen bottle for long-term preservation.

[0020] Example 2: Preparation of a double-stained transparent specimen of the skeleton of a bony fish (medium-sized, 7-15 cm in length) This embodiment details the optimized operating procedure for bony fish. (See also...) Figure 3 The method includes the following steps: 1. Sample preprocessing Take one grass carp (boneless fish), about 12 cm in length. Skin, gut, and clean it as described above. 2. Alcohol gradient fixation (1) Fixative: Prepare 80 mL of 50% ethanol using the 95% ethanol in the kit; (2) Gradient fixation: The procedure is the same as in Example 1, and the total fixation time is 5 days based on the optimization parameters; (3) Drying treatment: After fixation, the sample is taken out and placed in a 43℃ oven for gentle drying for 12 minutes to remove excess alcohol from the surface; 3. KOH pre-transparency (1) Transparent liquid: Weigh 2.5 g of KOH solid, dissolve it in 250 mL of distilled water, and prepare a 1% KOH solution; (2) Processing time: 4 hours according to the optimized parameters; 4. Cartilage staining (alicin blue staining) (1) Dye solution: Take 2.5 mL of Alcian Blue stock solution, add 200 mL of 95% ethanol and 47.5 mL of glacial acetic acid solution, mix well to prepare 250 mL of working solution. Adjust the pH of the dye solution to between 4.2 and 5.8 with KOH solution; (2) Processing time: According to the optimized parameters, staining time is 22 hours; 5. Cartilage decolorization (1) Decolorizing solution: Use 80 mL of 95% alcohol; (2) Processing time: According to the optimized parameters, decolorization takes 30 hours (the solution is changed every 8 hours); 6. Re-transparency (KOH becomes transparent again) (1) Re-transparent solution: Same as pre-transparent solution, prepare 250 mL of 1% KOH solution; (2) Processing time: 6 hours according to the optimized parameters; 7. Staining of bone (Alizarin Red staining) (1) Dye solution: Take 2.5 mL of alizarin red mother liquor, add 247.5 mL of distilled water and 2.5 g of KOH, mix and prepare 250 mL of working solution; (2) Processing time: According to the optimized parameters, staining time is 20 hours; 8. Bone decolorization (1) Decolorizing solution: Use 200 mL of distilled water; (2) Processing time: According to the optimized parameters, decolorization takes 36 hours (the solution is changed every 6 hours); 9. Muscle recoloring and bleaching (1) Bleaching solution: Prepare 150 mL of 3% hydrogen peroxide solution (15 mL of 30% hydrogen peroxide + 135 mL of distilled water); (2) Process until the muscle is colorless and transparent; 10. Gradient Glycerin Clearing and Storage (1) Gradient transparency: Soak in 25%, 50%, and 75% glycerol (each prepared with 0.5% KOH, approximately 150 mL each) for 24 hours each. Then continue to clear in 75% glycerol until complete (approximately 5 days). (2) Seal in pure glycerol containing thymol.

[0021] Example 3: Preparation of double-stained transparent specimens of cartilaginous fish skeletons (large, 15-25 cm in length) 1. Sample preprocessing Take one large sturgeon (cartilaginous fish), approximately 20 cm in length. Prepare it as described above. 2. Alcohol gradient fixation (1) Fixative: Prepare 120 mL of 50% ethanol using 95% ethanol from the kit; (2) Gradient fixation: The procedure is the same as before, and the total fixation time is 8 days based on the optimization parameters; (3) Drying treatment: After fixation, the sample is taken out and placed in a 45℃ oven for gentle drying for 15 minutes to remove excess alcohol from the surface; 3. KOH pre-transparency (1) Transparent liquid: Weigh 5 g of KOH solid, dissolve it in 500 mL of distilled water, and prepare a 1% KOH solution; (2) Processing time: 6 hours according to the optimized parameters; 4. Cartilage staining (alicin blue staining) (1) Dye solution: Take 5 mL of Alcian Blue stock solution, add 400 mL of 95% ethanol and 95 mL of acetic acid, mix well to prepare 500 mL of working solution. Adjust the pH of the dye solution to between 4.2 and 5.8 with KOH solution; (2) Processing time: According to the optimized parameters, staining time is 28 hours; 5. Cartilage decolorization (1) Decolorizing solution: Use 120 mL of 95% alcohol; (2) Processing time: According to the optimized parameters, decolorization takes 48 hours (the solution is changed every 4 hours); 6. Re-transparency (KOH becomes transparent again) (1) Re-transparent solution: Same as pre-transparent solution, prepare 500 mL of 2% KOH solution; (2) Processing time: 6 hours according to the optimized parameters; 7. Staining of bone (Alizarin Red staining) (1) Dye solution: Take 5 mL of alizarin red mother liquor, add 495 mL of distilled water and 5 g of KOH, mix and prepare 500 mL of working solution; (2) Processing time: According to the optimized parameters, staining time is 32 hours; 8. Bone decolorization (1) Decolorizing solution: Use 300 mL of distilled water; (2) Processing time: According to the optimized parameters, decolorization takes 48 hours (the solution is changed every 5 hours); 9. Muscle recoloring and bleaching (1) Bleaching solution: Prepare 200 mL of 3% hydrogen peroxide solution (20 mL of 30% hydrogen peroxide + 180 mL of distilled water); (2) Process until the muscle is colorless and transparent; 10. Gradient Glycerin Clearing and Storage (1) Gradient transparency: Soak in 25%, 50%, and 75% glycerol (each prepared with 0.5% KOH, approximately 200 mL each) for 24 hours each. Then continue to clear in 75% glycerol until complete (approximately 7 days). (2) Seal in pure glycerol containing thymol.

[0022] Summarize The above embodiments demonstrate in detail how to apply the quantitative parameter system and reagent kit of the present invention to perform standardized operations on fish specimens of different species and body sizes. By following the specific steps and parameters described above, high-quality skeletal specimens with bright blue cartilage, clear red bone, and transparent muscle tissue can be obtained stably and efficiently, fully verifying the practicality, reliability, and superiority of the present invention.

Claims

1. A method for preparing a double-stained transparent specimen of fish skeleton, characterized in that, Includes the following steps: S1. Sample pretreatment: Remove the skin and internal organs of the fish while keeping the skeletal structure intact; S2. Alcohol gradient fixation: The samples were sequentially fixed using alcohol solutions of concentrations of 50%, 75%, and 95%. S3, KOH pre-clearing: The sample is treated with KOH solution to make the muscle tissue semi-transparent; S4. Cartilage staining: Cartilage tissue was stained with Alcian blue staining solution; S5. Cartilage decolorization: Decolorization treatment using 95% alcohol solution; S6. Re-transparency: The sample is re-transparently transparent using KOH solution; S7. Staining of bone tissue: Alizarin red staining solution was used to stain bone tissue; S8. Bone decolorization: Decolorization treatment using distilled water; S9. Muscle recoloring and bleaching: The sample was decolorized and bleached using a 3% hydrogen peroxide solution; S10, Gradient Glycerin Clearing: The samples were treated sequentially with 25%, 50%, and 75% glycerin gradient clearing solutions; S11. Preservation: Place the transparent sample in a preservation solution for preservation. In particular, the processing parameters of at least one step in steps S2-S11 are selected and determined from a pre-constructed multi-factor optimization parameter matrix database based on the skeletal type and body length classification of the target fish.

2. A method for constructing a parameter matrix database used in the method as described in claim 1, characterized in that, The steps include: S100: Fish are classified into two types of skeletons: cartilaginous fish and bony fish; S200: For each skeletal type, it is divided into at least three grades based on body length; S300: For each grade, design a multi-factor orthogonal experimental scheme with KOH concentration, treatment time and staining time as factors; S400: Perform the orthogonal experiment to obtain samples under each experimental combination and measure their staining effect, muscle transparency and tissue structure integrity. S500: Perform statistical analysis on the experimental results to determine the optimal parameter combination for each level, forming the parameter matrix database.

3. The preparation method according to claim 1, characterized in that, When the target fish is a bony fish, the parameter combination in steps S3-S7 can be selected from any of the following: Small fish: KOH pre-clearing concentration 10g / L, time 3-5h; cartilage staining 20-24h; destaining 12-16h; re-clearing time 5-6h; bone staining 16-20h; Medium-sized fish: KOH pre-clearing concentration 10g / L, time 4h; cartilage staining 22-26h; decolorization 24-28h; re-clearing time 8-10h; bone staining 18-22h; Large fish: KOH pre-clearing concentration 20g / L, time 8-12h; cartilage staining 28-32h; destaining 32-36h; re-clearing time 10-12h; bone staining 22-24h; The parameter combination is derived from the results of a multi-factor orthogonal experiment on bony fish.

4. The preparation method according to claim 1, characterized in that, When the target fish is a cartilaginous fish, the parameter combination in steps S3-S7 can be selected from any of the following: Small fish: KOH pre-clearing concentration 10g / L, time 2-4h; cartilage staining 14-18h; destaining 20-24h; re-clearing time 1-3h; bone staining 18-22h; Medium-sized fish: KOH pre-clearing concentration 10g / L, time 3-5h; cartilage staining 18-22h; decolorization 28-32h; re-clearing time 2-4h; bone staining 20-24h; Large fish: KOH pre-clearing concentration 10g / L, time 6-8h; cartilage staining 28-32h; destaining 44-48h; re-clearing time 6-8h; bone staining 28-32h; The parameter combination is derived from the results of a multi-factor orthogonal experiment on large cartilaginous fish.

5. The preparation method according to claim 1, characterized in that, The alnicotinic blue staining working solution used in step S4 is prepared by mixing 1 volume part of the mother solution with 79 volume parts of 95% ethanol and 20 volume parts of glacial acetic acid, and adjusting the pH of the mixture to 4.2-5.8 with KOH solution.

6. The preparation method according to claim 1, characterized in that, The alizarin red staining solution used in step S7 is obtained by diluting the alizarin red staining mother solution, which is a 100× concentrate containing 0.05g alizarin red in every 5 mL of mother solution; the alizarin red staining working solution is prepared by mixing 1 volume part of the mother solution with 99 volume parts of distilled water, and adding KOH solid until the final KOH mass-volume ratio is 1%.

7. The preparation method according to claim 1, characterized in that, The gradient glycerol clearing solution is composed of 25% glycerol, which is made by mixing glycerol and 0.5% KOH solution in a volume ratio of 1:3; 50% glycerol clearing solution is made by mixing glycerol and 0.5% KOH solution in a volume ratio of 1:1; and 75% glycerol clearing solution is made by mixing glycerol and 0.5% KOH solution in a volume ratio of 3:

1.

8. The preparation method according to claim 1, characterized in that, In the cartilage decolorization and bone decolorization steps, the decolorizing solution is replaced when it shows a clear dye color, and the replacement interval is 4-8 hours.

9. The preparation method according to claim 1, characterized in that, After alcohol gradient fixation, the sample is placed in an environment of 40-45℃ for gentle drying for 5-15 minutes to remove residual alcohol on the surface.

10. A kit for preparing double-stained transparent specimens of fish skeletons, characterized in that, include: (1) Key staining reagent unit: contains Alcian blue staining stock solution (100× concentrate) and Alizarin red staining stock solution (100× concentrate), wherein each 5 mL of Alcian blue staining stock solution (100× concentrate) contains 50 mg of Alcian blue and 5 mL of pure ethanol, and each 5 mL of Alizarin red staining stock solution (100× concentrate) contains 0.05 g of Alizarin red; (2) Basic treatment reagent unit: containing predetermined amounts of KOH solid, ethanol, glycerol, hydrogen peroxide, and glacial acetic acid; (3) Standardized guidance unit: includes an instruction manual with a reference table of operation parameters for each step for cartilaginous fish and bony fish, small fish, medium fish and large fish of different sizes. The parameter matrix database lookup table in the standardized guidance unit includes the optimal parameter combination as described in any one of claims 3-4 for small, medium and large bony and cartilaginous fish.

Images