Optic nerve paraffin section, preparation method and kit
By combining local marking of the optic nerve with a specific fixative in paraffin sectioning method, the problems of low cut-out rate and inaccurate localization of optic nerve paraffin sections have been solved, achieving high success rate and high integrity of optic nerve sections, thus improving the reliability of the research.
Patent Information
- Application Number
- CN202511473400.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-15
- Publication Date
- 2025-12-12
AI Technical Summary
The low cut-out rate of paraffin sections of the optic nerve, along with poor integrity and clarity, makes accurate localization difficult and affects the reliability of subsequent studies.
The optic nerve was locally marked using hematoxylin solution, fixed with Davidson's fixative and 10% buffered neutral formalin solution, and then subjected to gradient dehydration and clearing treatment. It was then embedded in a three-cylinder paraffin to ensure the visibility and structural integrity of the optic nerve.
It significantly improved the success rate and localization accuracy of optic nerve slices, as well as the integrity and clarity of the slices, increasing them from the traditional 60% to over 90%, thus ensuring the authenticity and reliability of experimental data.
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Figure CN121113635A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of animal tissue section, and particularly relates to an optic nerve paraffin section, a preparation method and a kit. BACKGROUND
[0002] In the field of neuroscience research, the optic nerve (ON) as a key component of the central nervous system (CNS) has unique anatomical and histological characteristics, which are irreplaceable for building disease models and conducting drug evaluation. Especially in drug toxicity research, optic nerve sectioning technology has become an important tool for evaluating the potential toxicity of drugs on the central nervous system, neuroprotective effects and nerve regeneration capacity.
[0003] However, due to the particularity of the structure of the optic nerve, the cutting rate of its paraffin section is very low (usually less than 60%), and the integrity and clarity are poor. The main reasons are:
[0004] 1. The contrast of the optic nerve and the surrounding tissue is low, and the structure of the optic nerve often cannot be clearly identified, which makes it difficult to accurately determine the position of the optic nerve during sectioning, resulting in cutting or not cutting out. Although there are some marking methods for the optic nerve at present, such as molecular marking, fluorescent marking, transgenic method and functional marking, there is still a lack of corresponding marking technology in the specific field of paraffin sectioning, which makes it difficult to guarantee the integrity and accurate positioning of the optic nerve, affecting the reliability of subsequent research.
[0005] 2. Due to the thinness and shortness of the optic nerve, such as the diameter of the mouse optic nerve being only 0.2-0.3mm and the length being about 5-7mm, it is easy to break, twist or be unclearly positioned during paraffin sectioning, which makes it difficult to guarantee the integrity of the sample.
[0006] Therefore, when conducting research on the optic nerve based on paraffin section, there are problems such as difficulty in guaranteeing the integrity of the sample, difficulty in identifying the structure and low accuracy of positioning. Therefore, the development of a paraffin sectioning technology suitable for the optic nerve is of great importance to improve the accuracy and reliability of optic nerve research. The realization of this goal will greatly promote our understanding of central nervous system diseases and the development of treatment strategies. SUMMARY
[0007] The application provides an optic nerve paraffin section, a preparation method and a kit, which significantly improves the visibility, positioning accuracy and structural integrity of the optic nerve by improving the paraffin sectioning method, and obtains an optic nerve paraffin section with better integrity and clarity.
[0008] The application adopts the following technical solutions:
[0009] In a first aspect, the present application provides a method for preparing an optic nerve paraffin section, comprising the following steps:
[0010] S1. Obtaining an eyeball, and placing the eyeball in a fixing solution for preliminary fixation;
[0011] S2. Finding the optic nerve, and marking the proximal end of the optic nerve locally using a hematoxylin solution;
[0012] S3. Taking a tissue block of the marked optic nerve, and performing gradient dehydration treatment, transparency treatment, and paraffin immersion treatment on the tissue block;
[0013] S4. Paraffin embedding the tissue block after paraffin immersion;
[0014] S5. Sectioning and HE staining the embedded block to obtain a paraffin section.
[0015] Preferably, the concentration of the hematoxylin solution is 0.5-1.5%, more preferably 1%.
[0016] Preferably, the fixing solution in step S1 is a Davidson’s fixative solution and a 10% buffered neutral formalin solution, and the preliminary fixation is performed in the Davidson’s fixative solution for 6-24 hours; then the tissue is transferred to the 10% buffered neutral formalin solution for continued fixation for 12-48 hours.
[0017] Preferably, the paraffin immersion treatment in step S3 is performed using three cylinders of paraffin in sequence.
[0018] Preferably, the melting points of the three cylinders of paraffin are all 56°C, and the soaking time in each cylinder of paraffin is 40-60 minutes.
[0019] Preferably, in step S4, the paraffin embedding is performed in a manner that the largest longitudinal face of the optic nerve is facing downward, so that the long axis direction of the optic nerve is parallel to the bottom surface of the embedding mold.
[0020] Preferably, in step S3, the gradient dehydration treatment uses ethanol with concentrations of 60%, 70%, 80%, 90%, 95% I, 95% II, anhydrous ethanol I, and anhydrous ethanol II in sequence, and the soaking time for each step is 15-30 minutes.
[0021] Preferably, the transparency treatment uses xylene or an alternative transparency agent, and is performed twice, with a treatment time of 3-10 minutes for each time.
[0022] In a second aspect, the present application provides a kit for the preparation method described above, comprising a hematoxylin solution, a Davidson's fixative solution, a 10% buffered neutral formalin solution, a gradient ethanol series, a clearing agent and paraffin. There is a certain synergy between the components, for example, the combination of hematoxylin labeling and gradient dehydration steps ensures that the label remains stable and easy to identify even after multiple chemical treatments; and the selection of different fixatives optimizes the effect of tissue preservation according to their respective characteristics.
[0023] In a third aspect, the present application provides an optic nerve section obtained by the preparation method described above.
[0024] By implementing the technical solutions described above, the present application has the following beneficial effects:
[0025] The present application uses a hematoxylin solution to label the optic nerve in the early stage, greatly improving the visibility and recognition of subsequent sectioning, and solving the problem of inaccurate positioning in traditional methods. This method significantly improves the success rate of optic nerve sectioning from less than 60% to more than 90%.
[0026] The present application uses the combined use of Davidson's fixative and 10% buffered neutral formalin solution to ensure good preservation of the tissue, the former is suitable for initial rapid fixation, and the latter is more suitable for long-term preservation of sample structure. The combination of the two can more effectively maintain the original morphology and structure of the optic nerve, laying a foundation for subsequent accurate sectioning.
[0027] Further, the present application uses gradient ethanol and a clearing agent to gradually replace the water in the tissue, and finally replaces it with paraffin of different gradients for embedding in turn, which ensures the integrity and stability of the tissue, avoids tissue damage caused by direct immersion in high-concentration solvents, and ensures the authenticity and reliability of experimental data. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 A mouse optic nerve paraffin section obtained in Example 1 of the present application;
[0029] Figure 2 A mouse optic nerve paraffin section obtained in Example 2 of the present application;
[0030] Figure 3 A mouse optic nerve paraffin section obtained in Comparative Example 1 of the present application;
[0031] Figure 4 A mouse optic nerve paraffin section obtained in Comparative Example 2 of the present application;
[0032] Figure 5The mouse optic nerve paraffin section obtained in Invention Comparative Example 3. DETAILED DESCRIPTION
[0033] The technical solutions of the present application will be described clearly and completely below with the aid of examples. Obviously, the described examples are only some of the examples of the present application, but not all. Based on the examples of the present application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0034] Reagent preparation method:
[0035] Hematoxylin staining solution: commercially available hematoxylin staining solution (Harris) BA4041.
[0036] 0.5% hydrochloric acid ethanol: take 300 ml of anhydrous ethanol and add to 100 ml of water, mix well, and 75% ethanol is obtained. Take 2 ml of concentrated hydrochloric acid, slowly add to 398 ml of 75% ethanol, mix well, and it is ready.
[0037] 0.5% dilute ammonia water: add 5 drops of concentrated ammonia water to 450 ml of water and mix well.
[0038] Davidson's fixative solution: DF004, Beijing Legen Biotechnology Co., Ltd.
[0039] Tissue fixative (containing 10% buffered neutral formalin solution): B0011-0002, Jiangsu Shetai Experimental Equipment Co., Ltd.
[0040] 60% ethanol: 60 ml of anhydrous ethanol is added to 40 ml of water, mixed well, and it is ready.
[0041] 70% ethanol: 70 ml of anhydrous ethanol is added to 30 ml of water, mixed well, and it is ready.
[0042] 80% ethanol: 80 ml of anhydrous ethanol is added to 20 ml of water, mixed well, and it is ready.
[0043] 90% ethanol: 90 ml of anhydrous ethanol is added to 10 ml of water, mixed well, and it is ready.
[0044] 95% ethanol: 95 ml of anhydrous ethanol is added to 5 ml of water, mixed well, and it is ready.
[0045] Example 1:
[0046] This example takes mouse optic nerve as an example to provide a preparation method of optic nerve paraffin section, which comprises the following steps:
[0047] S1. ICR mice were sacrificed using cervical dislocation method and eyeballs were quickly removed and fixed in Davidson’s fixative solution overnight (12h) and then transferred to 10% buffered neutral formalin solution for further fixation (24h).
[0048] S2. Optic nerve labeling:
[0049] 20 mouse optic nerves were removed from eyeballs and the proximal end of a portion of the optic nerves were labeled with a toothpick tip dipped in hematoxylin solution for easy localization later. The rest of the optic nerves were not labeled and were cut longitudinally into 2mm-4mm tissue blocks.
[0050] S3. Gradient dehydration:
[0051] Tissue blocks were sequentially placed in different concentrations (60%, 70%, 80%, 90%, 95% I, 95% II, anhydrous ethanol I and II) of ethanol solution for 20 minutes in each concentration to gradually reduce the water content in the tissue.
[0052] S4. Clearing treatment:
[0053] Tissue blocks after gradient dehydration were sequentially placed in 2 cylinders of clearing agent xylene for 5 minutes each to further prepare for wax immersion.
[0054] S5. Wax immersion:
[0055] Tissue blocks after clearing were sequentially transferred to 3 cylinders of paraffin (melting point 56°C) for 50 minutes each to ensure complete penetration of paraffin into the tissue.
[0056] S6. Paraffin embedding:
[0057] Paraffin embedding was performed in the longitudinal maximum plane to ensure the natural shape of the optic nerve.
[0058] S7. Paraffin sectioning:
[0059] Fine trimming of the tissue block was performed when it approached the hematoxylin labeled site to prepare 3pm thick sections.
[0060] S8. HE staining:
[0061] After the slice is baked at 65℃ for 10 minutes, it is sequentially subjected to xylene I, xylene II transparency for 8 minutes, anhydrous ethanol I, anhydrous ethanol II for 5 minutes each, 90% ethanol, 80% ethanol I, 70% ethanol for 3 minutes each, tap water flushing for 3 minutes, hematoxylin staining for 7 minutes, tap water flushing for 3 minutes, hydrochloric acid ethanol differentiation for 8 seconds, tap water flushing for 3 minutes, dilute ammonia blueing for 1 minute, tap water flushing for 2 minutes, 80% ethanol II, 95% ethanol I for 3 minutes each, eosin staining for 1 minute, 95% ethanol II, 95% ethanol III for 80 seconds each, anhydrous ethanol III, anhydrous ethanol IV for 5 minutes each, xylene III for 10 minutes, and finally, the slice is dried and observed.
[0062] S9. Observation under a microscope:
[0063] After all the above steps are completed, the slice result is observed under a microscope, and the result is shown in Table 1. Figure 1 The optic nerve structure positioning is clear, and the retina and choroid are complete and clear in structure and do not fall off.
[0064] Example 2:
[0065] The difference between this example and Example 1 is that the fixing time in step S1 is adjusted, and the eyeball is fixed in Davidson’s fixative solution overnight (8h), and then transferred to 10% buffered neutral formalin solution for continuous fixation (48h). The slice result is observed under a microscope, and the result is shown in Table 2. Figure 2 The optic nerve positioning is clear, and the retina and choroid are complete and do not fall off under a microscope.
[0066] Comparative Example 1:
[0067] The difference from Example 1 is that Comparative Example 1 does not perform S2, that is, the optic nerve is not labeled with hematoxylin solution. The slice result is observed under a microscope, and the result is shown in Table 3. Figure 3 The optic nerve positioning is not clear, and it is not visible under a microscope.
[0068] Comparative Example 2:
[0069] The difference from Example 2 is that Comparative Example 2 does not perform S2, that is, the optic nerve is not labeled with hematoxylin solution. The slice result is observed under a microscope, and the result is shown in Table 4. Figure 4 The optic nerve positioning is not clear, and it is not visible under a microscope.
[0070] Comparative Example 3:
[0071] The difference from Example 1 is that in step S1, the eyeball is directly placed in 10% buffered neutral formalin solution for fixation for 48 hours. The slice result is observed under a microscope, and the result is shown in Table 5. Figure 5 The retina and choroid fall off.
[0072] Based on the results of the above embodiments and comparative examples, the first step in the technical solution of the present invention, which is to mark the optic nerve with hematoxylin solution, is one of the key steps. This step determines whether the subsequent optic nerve localization is clear and visible. Secondly, on this basis, the synergistic fixation with Davidson's fixative solution and 10% buffered neutral formalin solution also plays an important role in whether the optic nerve in the slice is clear and intact and whether the retina is detached.
[0073] The foregoing general description of the invention and its specific embodiments should not be construed as a limitation on the technical solution of the invention. Those skilled in the art, based on the disclosure of this application, can add, reduce, or combine the disclosed technical features in the foregoing general description and / or specific embodiments (including examples) without departing from the constituent elements of the invention, to form other technical solutions within the scope of protection of this application.
Claims
1. A method for preparing paraffin sections of the optic nerve, characterized in that, Includes the following steps: S1. Obtain the eyeball and place it in a fixative solution for initial fixation; S2. Locate the optic nerve and use hematoxylin solution to locally mark the proximal end of the optic nerve; S3. The marked optic nerve tissue blocks are then subjected to sampling, gradient dehydration, clearing, and paraffin impregnation. S4. Embed the paraffin-impregnated tissue blocks in paraffin; S5. Section the embedded block and stain it with hematoxylin and eosin (HE) to obtain paraffin sections.
2. The method for preparing paraffin sections of the optic nerve according to claim 1, characterized in that, The concentration of the hematoxylin solution is 0.5% to 1.5%.
3. The method for preparing paraffin sections of the optic nerve according to claim 1, characterized in that, The fixative in step S1 is Davidson's fixative solution and 10% buffered neutral formalin solution. Initial fixation is performed in Davidson's fixative solution for 6-24 hours. The tissue was then transferred to a 10% buffered neutral formalin solution for further fixation for 12–48 hours.
4. The method for preparing paraffin sections of the optic nerve according to claim 1, characterized in that, In step S3, the wax impregnation process is carried out sequentially using three cylinders of paraffin wax.
5. The method for preparing paraffin sections of the optic nerve according to claim 1, characterized in that, The three cylinders of paraffin wax all have a melting point of 56℃, and the soaking time in each cylinder of paraffin wax is 40-60 minutes.
6. The method for preparing paraffin sections of the optic nerve according to claim 1, characterized in that, In step S4, the paraffin embedding is performed with the longitudinal maximum surface facing downwards, so that the long axis of the optic nerve is parallel to the bottom surface of the embedding mold.
7. The method for preparing paraffin sections of the optic nerve according to claim 1, characterized in that, In step S3, the ethanol concentrations used in the gradient dehydration treatment are 60%, 70%, 80%, 90%, 95% I, 95% II, anhydrous ethanol I, and anhydrous ethanol II, respectively, and the soaking time for each step is 15-30 minutes.
8. A kit for use in the preparation method according to any one of claims 1-7, characterized in that, It contains hematoxylin solution, Davidson's fixative solution, 10% buffered neutral formalin solution, a gradient ethanol series, a clearing agent, and paraffin.
9. A paraffin section of the optic nerve, characterized in that, It is obtained by the preparation method described in any one of claims 1-8.