Kit and method suitable for Golgi staining of large animals

Abstract

The invention relates to a kit and a method suitable for Golgi staining of large animals. The kit comprises: 1) an impregnation liquid, wherein the impregnation liquid is an AB liquid, and the AB liquid is an isovolumetric mixed liquid of 5 wt% potassium dichromate (liquid A) and 5 wt% mercuric chloride (liquid B); the solution C is 1.4 wt% potassium chromate solution, the color developing solution is a mixed solution of a solution D, a solution E and flushing fluid in a volume ratio of 1: 1: 1.8-1: 1: 2.5, and the solution D is a mixed solution of 25vol% stronger ammonia water and Milli-Q water; the solution E is a sodium thiosulfate pentahydrate aqueous solution; and the flushing fluid is Milli-Q water. Methods of Golgi staining of large animal brain tissue are also provided. According to the method, high-definition and quantitative-analysis staining results are provided for neuroscience research of the domestic dogs, and reference is provided for optimization of Golgi staining technologies of other large animals.

Description

Technical Field

[0001] This invention belongs to the field of pathology technology and relates to kits and methods for Golgi staining of large animals. Background Technology

[0002] Golgi staining, first proposed by Camillo Golgi in 1873, utilizes the metathesis reaction of potassium dichromate and silver nitrate to generate silver chromate precipitate. Taking advantage of the argyrophilic properties of neurons, this precipitate is selectively deposited on the cell body and processes, thus revealing the complete neuronal morphology under a light microscope. This method can reveal changes in dendritic spine morphology (such as dendritic atrophy in Alzheimer's disease), synaptic plasticity, and neurodevelopmental abnormalities, making it an important tool in neuropathological research.

[0003] In rodents (such as mice and rats) widely used in neuroscience research, Golgi staining has been gradually replaced by in vivo immunofluorescence labeling due to its long cycle (14-21 days of immersion), low tolerance for errors (uneven penetration leading to high background noise and blurred neuronal boundaries), and toxicity risks (reagents such as silver nitrate and mercury salts). However, in large animals such as domestic dogs that exhibit advanced human social behaviors and have complex brain tissue characteristics, in vivo immunofluorescence is difficult to widely apply due to poor blood-brain barrier penetration and high systemic administration costs. Golgi staining remains the primary method for studying neuronal maturity. However, the application of Golgi staining directly transplanted from rodents in canine brain research faces a series of unresolved challenges, such as: the traditional 14-day immersion cycle cannot penetrate the high-density brain tissue of domestic dogs; the staining depth is uneven due to unsuitable immersion parameters; the conventional 100-150 μm section thickness easily causes tissue fragmentation in highly myelinated areas of domestic dogs; and the lipid content of canine brain tissue is significantly higher than that of rodents. Therefore, developing a standardized Golgi staining procedure suitable for domestic dogs is particularly necessary.

[0004] Based on the above-mentioned deficiencies, this invention makes the following targeted optimizations to the characteristics of canine brain tissue: improve the impregnation and penetration efficiency of canine brain tissue, find a suitable section thickness for canine brain tissue, improve the section permeation quality while using a more environmentally friendly and safe dewaxing agent, and improve a Golgi staining process suitable for canine brain tissue that can obtain clear and statistically significant neuronal images in the brain tissue of dogs and even large animals. Summary of the Invention

[0005] The technical objective of this invention is to provide an optimized method and related reagent kit for Golgi staining of large animal brain tissue. By coordinating anesthesia and perfusion control, adjusting gradient immersion parameters, improving sectioning techniques, and standardizing the staining process, this invention solves the problems of insufficient staining penetration, high background noise, and blurred dendritic spine morphology in existing technologies for large animal brain tissue. It provides high-resolution and quantitatively analyzable staining results for large animal neuroscience research and provides a reference framework for optimizing other large animal Golgi staining techniques.

[0006] To achieve the above-mentioned technical objectives, on the one hand, this invention provides a kit for Golgi staining of brain tissue in large animals, the kit comprising: 1) Impregnation solution AB solution: An equal volume mixture of 5 wt% potassium dichromate (solution A) and 5 wt% mercuric chloride (solution B); Solution C: 1.4 wt% potassium chromate solution. 2) Color developer The colorimetric solution is a mixture of solution D:solution E:rinsing solution in a volume ratio of 1:1:1.8 to 1:1:2.5. in, Solution D: 25 vol% concentrated ammonia solution and Milli-Q water are mixed in a volume ratio of 3:1; Solution E: 1.57 g sodium thiosulfate pentahydrate / 100 mL Milli-Q water; Rinse solution: Milli-Q water.

[0007] In a specific implementation, the large animal is selected from domestic pigs, domestic dogs, sheep, cattle, and monkeys, with domestic dogs being the preferred choice.

[0008] On the other hand, the present invention provides a method for Golgi staining of large animal brain tissue using the above-mentioned kit, the method comprising the following steps: 1) Fixed: Immerse isolated brain tissue blocks from large animals in the above-mentioned AB solution pre-cooled to 4°C and store them in the dark for 1 day for fixation. 2) Pre-impregnation: The fixed tissue block was immersed in the pre-cooled AB solution and stored in a light-protected environment at 4℃-8℃ for 1 day. 3) Main impregnation: Soak the tissue blocks in the above AB solutions at room temperature in the dark for 14 days; then replace with the above C solution and soak in the dark for 3-4 days. 4) Frozen slices After freezing the tissue block from step 3), section it, and set the section thickness to 80 μm. 5) Color development The sections were developed using the above-mentioned colorimetric solution; 6) Imaging statistics.

[0009] In a specific implementation, the volume of the isolated brain tissue block from a large animal is 1 cm³. 3 .

[0010] In a specific implementation, the pre-cooling temperature in step 2) is 4°C.

[0011] In a specific implementation, in step 4), the tissue block from step 3) is pre-cooled at -22°C for 4 hours and then sliced.

[0012] In a specific implementation, step 5) includes the following procedures: 5-1) Rinse: Rinse the sections twice with Milli-Q water, 4 minutes each time; 5-2) Color development: Immerse the staining vessel in the freshly prepared color developing solution for 10 minutes, gently shaking it every 2 minutes during this time. 5-3) Dehydration and Transparency: Dehydration was carried out sequentially through a gradient of 50 vol % → 75 vol % → 95 vol % ethanol, each for 4 minutes; Next, dehydrate with anhydrous ethanol four times, each time for no more than four minutes; Finally, use an environmentally friendly dewaxing agent to clear the wax three times, with the first two times for 4 minutes and the last time for 6 hours. 5-4) Covering: Add a drop of neutral resin mounting medium, cover with a coverslip, and store at room temperature away from light.

[0013] In a specific implementation, step 6) includes: Using a laser scanning confocal microscope, uniformly stained areas were selected under a 10× objective lens. Then, Z-stack layer scanning was performed at a 100× oil immersion lens with NA=1.4, with a layer thickness of 0.5 μm. Images were saved as 16-bit TIFF format. Using ImageJ software, the "Multi-Point" tool was used to manually label slender, mushroom-shaped, short and stout, and filamentous pseudopodia-type dendritic spines. A 200×200 μm section was randomly selected from each slice. 2 Statistics on 5 visual fields.

[0014] In a specific implementation, the large animal is selected from domestic pigs, domestic dogs, sheep, cattle, and monkeys, with domestic dogs being the preferred choice.

[0015] Beneficial effects This application utilizes a dog-specific anesthesia-perfusion combination protocol (Zoletil + propofol + 100 mmHg pressure) to ensure tissue viability.

[0016] Furthermore, this application employs dynamic gradient control: by using a gradient impregnation strategy of AB mixture → C solution, combined with gentle mechanical swirling (violent shaking is prohibited), the uniformity of reagent penetration is improved.

[0017] Furthermore, considering the characteristics of high myelination and high lipid content in canine brain tissue, this application adjusts the immersion time, section thickness, and chromogenic solution ratio to overcome the penetration limitations of traditional rodent staining methods.

[0018] In summary, through the above improvements, this application can shorten the staining cycle to 18 days (compared to 21-28 days using traditional methods), achieving a section integrity rate of 95%. Dendritic spine clarity is improved, and background noise is reduced by 37%. In addition, the environmentally friendly color development and dehydration process in this application (optimized D / E liquid ratio + non-toxic dewaxing agent) balances effectiveness and safety.

[0019] Therefore, the Golgi body staining method of this application solves the problems of insufficient staining penetration, high background noise, and blurred dendritic spine morphology in the existing technology in canine brain tissue, providing high-definition and quantitatively analyzable staining results for canine neuroscience research, and providing a reference for the optimization of Golgi staining techniques in other large animals. Attached Figure Description

[0020] Figure 1 The results observed in canine brain tissue using conventional Golgi staining are shown.

[0021] A: Image observed under a 10X objective lens of a laser confocal microscope; B: Image observed under a 100X objective lens of a laser confocal microscope.

[0022] Figure 2 The results observed in canine brain tissue using the improved Golgi staining method described in this application are shown.

[0023] A: Image observed under a 10X objective lens of a laser confocal microscope; B: Image observed under a 100X objective lens of a laser confocal microscope. Detailed Implementation

[0024] The technical solutions of this application are described in detail below through specific embodiments to enable those skilled in the art to better understand this application. However, these embodiments are not intended to limit the scope of protection of this application.

[0025] Comparative example: (1) Place the canine brain tissue into pre-cooled Golgi stain (a mixture of potassium dichromate and silver nitrate) and let it stand at 4°C in the dark. The staining time is relatively long, generally 2-4 weeks. During this period, the staining solution needs to be changed regularly (every 3-5 days) to ensure that silver ions fully penetrate into the neurons.

[0026] (2) After the staining is completed, the tissue is washed with distilled water to remove the residual staining solution on the surface, and then transferred to a 30% sucrose solution to dehydrate and harden until the tissue settles to the bottom (1-2 days).

[0027] (3) After hardening, remove the tissue, gently dry it with absorbent paper, and embed it with Tissue Freezing Medium. Slice the tissue using a frozen section or a vibratory microtome to a thickness of 100-200 μm to preserve the axons and dendrites of neurons intact.

[0028] (4) The slides are stained a second time with silver nitrate solution (e.g., 0.75% concentration) under light-protected conditions to allow silver ions to be deposited in the neurons and form visible brown-black precipitates. This step takes about 1-2 days.

[0029] (5) The sections were dehydrated in sequence with 70 vol%, 95 vol%, and 100 vol% ethanol to remove water; then cleared with xylene to enhance tissue transparency.

[0030] (6) Add neutral resin or Canada balsam, cover with a coverslip, and let it air dry at room temperature before observing it through an optical microscope.

[0031] The results are as follows Figure 1 As shown in the image.

[0032] Example 1: Collection and Preprocessing of Canine Brain Tissue Materials and reagents: The AB solution for the Golgi kit: Biolead's FD Rapid GolgiStain™ Kit (large) Golgi kit.

[0033] Preparation of 100ml Golgi staining AB solution: Solution A is 50ml distilled water + 2.5g potassium dichromate, dissolved using a magnetic stir bar to prepare a 5% potassium dichromate solution; Solution B is 50ml distilled water + 2.5g mercuric chloride, dissolved using a magnetic stir bar to prepare a 5% mercuric chloride solution. Mix the two solutions in equal volumes and store at room temperature away from light for later use.

[0034] (1) Anesthesia and perfusion Zoletil 50 (a combination anesthetic of telatamine and zoprazepam, purchased from Roman Animal Health Products Co., Ltd.) was administered intramuscularly at a dose of 7-25 mg / kg to induce anesthesia, ensuring that the dogs (purchased from the National Canine Laboratory Animal Resource Bank) quickly entered a state of deep anesthesia to avoid the impact of stress on brain tissue. Whole-body perfusion was performed using 0.9% saline (purchased from Beyotime Biotechnology Co., Ltd.), with the perfusion pressure set at 100 mmHg and the perfusion fluid temperature maintained at 37°C until the outflow became clear (approximately 30 minutes), thoroughly removing residual blood and metabolic products from the blood vessels.

[0035] Compared with traditional single anesthesia (such as sodium pentobarbital), this application can avoid hypoxic brain tissue damage by using the above combination of anesthetics and dosage control, while reducing the interference of residual anesthetics on subsequent staining.

[0036] Furthermore, in the perfusion process, this application sets the perfusion pressure to 100 mmHg (traditional methods often use 80-120 mmHg randomly set), combined with 37℃ constant temperature physiological saline. Experiments have shown that this parameter can improve the microvascular permeability of canine brain tissue and reduce background staining caused by residual red blood cells.

[0037] (2) Fixation was performed using tissue fixative. Rapid fixation with AB solution: The trimmed brain tissue block (1 cm) 3 Immediately immerse it in the above AB solution pre-cooled to 4°C, and store it in a 4°C environment away from light.

[0038] Compared with traditional methods that use only a single fixative, this application uses AB solution for fixation, which shortens the stabilization time of neuronal membrane structure through the synergistic effect of metal ions.

[0039] In addition, this embodiment also tested that the AB solution of the Golgi reagent kit can also be used for Golgi staining of other large animals.

[0040] Example 2: Gradient Impregnation and Slicing Process Materials and reagents: The reagents are prepared as follows: The 100ml Golgi staining AB solution was the same as in Example 1.

[0041] Tissue Freezing Medium, gelatin-coated microscope slides, and Golgi staining solution C were purchased from Biolead. Golgi staining solution AB: purchased from Biolead's FD Rapid GolgiStain™ Kit (large) Golgi kit.

[0042] Golgi staining solution C: 1.4 wt% potassium chromate solution.

[0043] (1) Three-stage impregnation strategy Stepwise permeation control: The tissue blocks fixed in Example 1 were subjected to a gradient treatment of "primary immersion in AB solution for 14 days (gently rotate horizontally each day, do not shake violently) → enhanced treatment in C solution for 3-4 days" (the traditional method is a single immersion for 21 days). Experiments showed that this strategy significantly improved the uniformity of reagent permeation while avoiding mechanical damage to the tissue.

[0044] (2) Optimization of frozen section parameters Low-temperature sectioning anti-crystallization technique: After impregnation, use Tissue Freezing Medium to fix the bottom of the tissue block on the ice-cut sample holder, and perform sectioning under the condition of pre-cooling at -22℃ for 4 hours (the traditional method is mostly -18℃). The section thickness is set to 80 μm (domestic dogs have thicker myelin sheaths, and traditional 100-200 μm sections are prone to uneven staining). Use sterile plastic forceps to transfer the sample to a glass slide coated with gelatin containing Golgi staining C solution. After air drying at room temperature in the dark for 1 day, the sections can be stored for 3 days.

[0045] Example 3: Color Development Process Materials and reagents: Golgi staining D / E solution: purchased from Biolead's FD Rapid GolgiStain™ Kit (large).

[0046] Environmentally friendly dewaxing and clarifying agent: purchased from Solarbio.

[0047] (1) Rinse Rinse the slices from Example 2 twice with freshly prepared double-distilled water or Milli-Q water for 4 minutes each time.

[0048] (2) Color development Colorimetric working solution formulation: Solution D: 25% concentrated ammonia solution and Milli-Q water are mixed in a 3:1 ratio; Solution E: 1.57 g sodium thiosulfate pentahydrate dissolved in 100 mL Milli-Q water; Rinse solution: Milli-Q water, The colorimetric working solution is prepared by mixing solution D, solution E, and rinsing solution in a volume ratio of 1:1:2 (1:1:1 in the traditional method).

[0049] After rinsing, the slides were placed in the freshly prepared chromogenic working solution and incubated for 10 minutes. During this time, the staining vessel was gently shaken every 2 minutes to agitate the working solution and ensure full contact between the slides and the working solution. Experiments confirmed that a D / E solution and water in a 1:1:2 volume ratio can reduce excessive deposition of mercury salts in high-lipid regions of canine brain tissue, significantly improving the clarity of dendritic spine boundaries.

[0050] (3) Environmentally friendly dehydration and transparency process (3.1) Rinse the sections twice with freshly prepared double-distilled water or Milli-Q water for 4 minutes each time. Then dehydrate the sections in 50 vol%, 75 vol%, and 95 vol% ethanol for 4 minutes at each concentration gradient. Then dehydrate the sections in anhydrous ethanol for 4 times, each time for no more than 4 minutes. Use an environmentally friendly dewaxing agent instead of xylene for 3 gradient clearings (4 minutes for the first two times and 6 hours for the last time). Compared with traditional xylene, the toxicity is reduced by 90% and there is no difference in tissue transparency.

[0051] (3.2) After clearing, use absorbent paper to remove the residual environmentally friendly dewaxing and clearing agent on the slides, and place the tissue slides at room temperature in the dark for 15 minutes. When the tissue begins to turn white, start mounting.

[0052] (4) Sealing Use a neutral resin mounting medium to mount the slide. Generally, one drop of the mounting medium is sufficient. Before the mounting medium dries, place the coverslip on the slide to complete the mounting. Store at room temperature away from light and photograph it with a microscope as soon as possible.

[0053] Example 4: Standardization of Imaging and Data Analysis (1) Confocal microscopy The stained tissue sections were photographed using a laser confocal microscope (Olympus FV1000). The overall state of neurons was observed under a 10X objective. Then, the z-stack function of the laser scanning confocal microscope was used to photograph the required neuronal dendrites and dendritic spines under a 100X objective (NA=1.4), and the images were saved as 16-bit TIFF format images.

[0054] result( Figure 2 As shown in A and B), clear, statistically effective images of tissue sections can be obtained under both 10X and 100X objectives.

[0055] (2) Image analysis Experimenters not involved in the experiment manually labeled slender, mushroom-shaped, short and thick, and filamentous pseudopodia-type dendritic spines using ImageJ software (version v1.53t) and the "Multi-Point" tool. Five fields of view (200×200 μm) were randomly selected from each slice. 2)Statistical analysis was conducted using independent double-blind experiments.

[0056] from Figure 2 As can be seen from the images, dendritic spines exhibit four morphologies: slender, mushroom-shaped, short and stout, and filopodia. The slender and filopodia types are considered immature, while the short and stout and mushroom-shaped types are mature.

[0057] The results observed in canine brain tissue using the traditional Golgi staining method ( Figure 1 Compared to traditional Golgi staining, the improved Golgi body staining method of this application improves the dendritic spine sharpness score and reduces background noise by 37%. Background noise calculation: Pure background areas (such as unstained blank areas) with no obvious structure are selected in the images of traditional Golgi staining and the improved Golgi staining method of this application, and the standard deviation of the gray values ​​of all pixels in the area is calculated.

Claims

1. A kit for Golgi staining of brain tissue in large animals, the kit comprising: 1) Impregnation solution AB solution: An equal volume mixture of 5 wt% potassium dichromate (solution A) and 5 wt% mercuric chloride (solution B); Solution C: 1.4 wt% potassium chromate solution. 2) Color developer The colorimetric solution is a mixture of solution D:solution E:rinsing solution in a volume ratio of 1:1:1.8 to 1:1:2.

5. in, Solution D: 25 vol% concentrated ammonia solution and Milli-Q water are mixed in a volume ratio of 3:1; Solution E: 1.57 g sodium thiosulfate pentahydrate / 100 mL Milli-Q water; Rinse solution: Milli-Q water.

2. The kit according to claim 1, wherein, The large animals are selected from domestic pigs, domestic dogs, sheep, cattle, and monkeys, with domestic dogs being the preferred choice.

3. A method for Golgi staining of large animal brain tissue using the kit as described in claim 1, the method comprising the following steps: 1) Fixed: Immerse isolated brain tissue blocks from large animals in the above-mentioned AB solution pre-cooled to 4°C and store them in the dark for 1 day for fixation. 2) Pre-impregnation: The fixed tissue block was immersed in the pre-cooled AB solution and stored in a light-protected environment at 4℃-8℃ for 1 day. 3) Main impregnation: Soak the tissue block in the AB solution at room temperature in the dark for 14 days; then replace with the C solution and soak in the dark for 3-4 days. 4) Frozen slices After freezing the tissue block from step 3), section it, and set the section thickness to 80 μm. 5) Color development The slides were developed using the aforementioned developing solution; 6) Imaging statistics.

4. The method according to claim 3, wherein, The large animals are selected from domestic pigs, domestic dogs, sheep, cattle, and monkeys, with domestic dogs being the preferred choice.

5. The method according to claim 4, wherein, The volume of the isolated brain tissue block from a domestic dog is 1 cm. 3 .

6. The method according to claim 4, wherein, In step 2), the pre-cooling temperature is 4°C.

7. The method according to claim 4, wherein, In step 4), the tissue block from step 3) is pre-cooled at -22°C for 4 hours and then sliced.

8. The method according to claim 4, wherein, Step 5) includes the following procedures: 5-1) Rinse: Rinse the sections twice with Milli-Q water, 4 minutes each time; 5-2) Color development: Immerse the staining vessel in the freshly prepared color developing solution for 10 minutes, gently shaking it every 2 minutes during this time. 5-3) Dehydration and Transparency: Dehydration was carried out sequentially through a gradient of 50 vol % → 75 vol % → 95 vol % ethanol, each for 4 minutes; Next, dehydrate with anhydrous ethanol four times, each time for no more than four minutes; Finally, use an environmentally friendly dewaxing agent to clear the wax three times, with the first two times for 4 minutes and the last time for 6 hours. 5-4) Covering: Add a drop of neutral resin mounting medium, cover with a coverslip, and store at room temperature away from light.

9. The method according to claim 4, wherein, Step 6) includes: Using a laser scanning confocal microscope, uniformly stained areas were selected under a 10× objective lens. Then, Z-stack layer scanning was performed at a 100× oil immersion lens with NA=1.4, with a layer thickness of 0.5 μm. Images were saved as 16-bit TIFF format. Using ImageJ software, the "Multi-Point" tool was used to manually label slender, mushroom-shaped, short and stout, and filamentous pseudopodia-type dendritic spines. A 200×200 μm section was randomly selected from each slice. 2 Statistics on 5 visual fields.

Images