Manufacturing method of continuous pathological section of whole digestive tract of animal

Abstract

The invention discloses a method for manufacturing a continuous pathological section of an animal whole digestive tract, which relates to the technical field of pathological examination, and ensures that a fixing agent can fully permeate into the inner and outer walls of the digestive tract and a mucous membrane plica structure through pre-fixing treatment and shaping treatment on a tissue structure of the animal whole digestive tract; therefore, the mucous membrane layer is effectively prevented from being separated from the lower tissue, and the probability of tissue fracture and deformation is greatly reduced; meanwhile, the curling problem of the esophagus, the stomach and the intestinal tract in the dehydration and embedding process can be effectively solved, and high-precision flattening and shaping of the digestive tract are achieved; according to the manufacturing method, the unfolding and shaping quality of the digestive tract tissue structure is remarkably improved, the continuity and integrity of the slice are guaranteed, falling, deformation or dislocation of the tissue is avoided, full-view-angle display is provided for observing the expansion path and rule of digestive tract lesions, and the application prospect is wide. The pathological change process of complicated digestive tract diseases (such as digestive tract inflammation, tumors and the like) can be revealed, and the method is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The invention relates to the technical field of pathological examination, and in particular to a method for preparing continuous pathological sections of the entire digestive tract of an animal. Background Art

[0002] Animal specimens refer to specimens that have been carefully prepared by humans to display the microscopic morphology of specific tissues. They are widely used in scientific research and teaching. Although the existing technology has rich experience in the preparation of conventional animal pathology sections, for some special animal tissues or organs (such as the entire digestive tract), traditional specimen preparation methods often fail to meet their unique morphological requirements, resulting in poor section quality and failure to accurately reflect their internal microstructure.

[0003] The digestive tract is an important organ for animals to ingest, digest, and absorb nutrients. There are significant differences in the tissue structure, function, and embryonic development characteristics of each part. Therefore, when conducting digestive tract pathology research, high-quality pathological sections are crucial, especially in continuous pathological sections from the esophagus to the stomach, small intestine, large intestine, etc., it is even more necessary to avoid the dislocation and deformation of each tissue and ensure the integrity of each tissue.

[0004] Especially in the preparation of slices from the esophagus to the stomach, due to the long tubular structure of the esophagus, there are significant tissue differences at the junction of the esophagus and the stomach (cardia area). The esophagus is composed of multiple layers of squamous epithelium, while the gastric glandular area is composed of columnar epithelium, and the stomach is divided into glandless and glandular areas with different digestive functions. Therefore, how to preserve the integrity of these tissues and avoid dislocation and deformation caused by differences in structure and function between the esophagus and stomach is one of the key issues in specimen preparation.

[0005] In the process of making traditional digestive tract slices, not only is the intestinal tract long and the cavity structure complex, but the structural differences from the esophagus to the stomach also pose a great challenge to the preparation of high-quality continuous slices. As a passage connecting the mouth and the stomach, the esophagus is a slender tubular tube mainly composed of multiple layers of squamous epithelium. Its tissue structure is relatively simple, and its main function is to mechanically transport food. However, the junction of the esophagus and the stomach, the cardia area, is an important anatomical transition point with significant histological differences. The esophageal squamous epithelium and the gastric columnar epithelium transition here. If it is not handled properly during the slice making process, it is easy to cause misalignment between tissues, damage to the interface structure and deformation of the morphology, especially the integrity of the transition area between the two is difficult to preserve.

[0006] The structure of the stomach is more complex, with glandular and glandless areas performing different digestive functions. The esophageal stomach is mainly composed of squamous epithelium, which is relatively wear-resistant, while the glandular stomach is rich in gastric glands, secretes digestive juices, and has chemical digestion functions. When making slices, the structural differences in the transition zone from the esophagus to the stomach and different areas inside the stomach often lead to dislocation or shedding of tissues during slice making. In particular, the glandular area, because it contains a large number of secretory cells, is easily mechanically damaged during fixation and slicing, affecting the continuity and integrity of the slices. In addition, the obvious change in the thickness of the stomach wall is also one of the important factors that lead to reduced slice quality, especially in the connection area between the esophageal stomach and the glandular stomach. Improper handling will lead to tissue deformation or dislocation.

[0007] It can be seen that ensuring the integrity and accuracy of each anatomical transition area in the digestive tract is not only the key to preparing complete continuous sections, but also a necessary condition to ensure the accuracy of pathological analysis. Therefore, when preparing continuous pathological sections from the esophagus to the stomach, it is necessary to optimize and improve the existing sectioning method in order to overcome the problem of poor sectioning quality caused by the cavity structure of the intestine itself, the transition structure between the esophagus and the stomach, and the tissue differences in different areas of the stomach. Summary of the invention

[0008] The purpose of the invention is to propose a method for preparing continuous pathological sections of the entire digestive tract of animals, in view of the problems that continuous pathological sections of the entire digestive tract of animals prepared by the existing digestive tract section preparation method have poor continuity and integrity, and tissue shedding, deformation or dislocation, which significantly improves the quality and observation effect of pathological sections of the entire digestive tract of animals.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing continuous pathological sections of the entire digestive tract of an animal comprises the following steps: S1: Dissect and obtain the entire digestive tract tissue structure of the animal; S2: Pre-fix and shape the entire digestive tract tissue structure of the animal; S3: The shaped animal's entire digestive tract tissue structure is sequentially fixed, dehydrated, transparentized, embedded, sliced, stained and sealed to obtain continuous pathological sections of the animal's entire digestive tract.

[0010] The present invention provides a method for preparing continuous pathological sections of the entire digestive tract of an animal. By pre-fixing and shaping the tissue structure of the entire digestive tract of the animal, it is not only ensured that the fixative can fully penetrate into the inner and outer walls of the digestive tract and the mucosal fold structure, thereby effectively preventing the separation of the mucosal layer and the underlying tissue, and greatly reducing the probability of tissue breakage and deformation. At the same time, it can also effectively improve the curling problem of the esophagus, stomach and intestines during dehydration and embedding, and achieve high-precision flattening and shaping of the digestive tract. The preparation method has good universality, high stability, and strong operability. It can significantly improve the unfolding and shaping quality of the digestive tract tissue structure, ensure the continuity and integrity of the slices, avoid the shedding, deformation or dislocation of the tissue, and provide a full-view display for observing the expansion path and laws of digestive tract lesions. It is conducive to revealing the pathological changes of complex digestive tract diseases (such as digestive tract inflammation, tumors, etc.), and is suitable for large-scale promotion and application.

[0011] Among them, in step S1, preferably, before dissecting and obtaining the entire digestive tract tissue structure of the animal, the animal to be dissected is fasted for at least 10 hours; this can promote intestinal emptying and facilitate the acquisition of a more complete and clean entire digestive tract tissue.

[0012] Preferably, during the process of dissecting and obtaining the entire digestive tract tissue structure of an animal, 3-5°C physiological saline is used for washing to remove blood stains, intestinal contents, digestive juices, etc., to prevent tissue damage.

[0013] Preferably, the entire digestive tract tissue structure includes the complete and continuous esophagus, stomach, duodenum, jejunum, colon, cecum, rectum and anus of an animal.

[0014] Among them, in step S2, the pre-fixation treatment can prevent the autolysis and corruption of tissues caused by digestive juices, so that cells and tissues maintain their original morphology and structure, providing sufficient time for the subsequent shaping treatment of the digestive tract.

[0015] Preferably, the pre-fixation treatment comprises: soaking the entire digestive tract tissue structure of the animal in a neutral formalin solution for 25-35 minutes.

[0016] Only under the optimal pre-fixation conditions can the tissue have a certain hardness and toughness, while not being too hard to affect the subsequent shaping, thus facilitating the subsequent organization and shaping of the digestive tract; soaking time that is too long will make the digestive tract more prone to curling and deformation during shaping.

[0017] Preferably, the pH value of the neutral formalin solution is 7.2-7.4; the volume concentration is 9-11%.

[0018] Preferably, the shaping process comprises: a. Unilaterally cut open the entire digestive tract tissue structure that has been pre-fixed along the digestive tract cavity, so that the entire digestive tract tissue structure can be unfolded along the cut position; b. The whole digestive tract tissue structure cut open on one side is unfolded and rolled into concentric rings to obtain the shaped whole digestive tract tissue structure of the animal.

[0019] Preferably, in step b, when winding into concentric rings, the inner wall of the unfolded digestive tract tissue structure is the inner circle and the outer wall is the outer circle, and the unfolded digestive tract tissue structure is ensured to be in the same plane when wound.

[0020] Preferably, in step b, a shaping device is used for winding and shaping; the shaping device includes a sorter, a winding rod and a limiting plate; the sorter is rotatably mounted on the winding rod; the limiting plate is located inside the sorter and is arranged on the winding rod through; the sorter is provided with a sorting groove for flattening the entire digestive tract tissue structure; the winding rod is provided with a fixing groove for clamping and fixing one end of the entire digestive tract tissue structure; the limiting plate includes a first limiting plate and a second limiting plate, which are used to ensure that the unfolded digestive tract tissue structure is in the same position when winding. The shaping device has a simple structure and is easy to operate. It can quickly realize the unfolding and winding of the digestive tract tissue structure, ensure that the fixative can fully penetrate the inner and outer walls of the digestive tract and the mucosal fold structure, prevent the separation of the mucosal layer from the underlying tissue, greatly reduce tissue breakage and deformation, and prevent the esophagus, stomach and intestine from curling during dehydration and embedding. It realizes high-precision flattening and shaping of the digestive tract, significantly improves the shaping efficiency and quality of the digestive tract tissue structure, and plays a positive role in ensuring the continuity and integrity of the slices and avoiding the shedding, deformation or dislocation of the tissue.

[0021] Preferably, a limiting protrusion is further provided on the winding rod to prevent the first limiting plate from sliding downward along the winding rod.

[0022] Preferably, the limiting plate is a filter paper board; while ensuring that the unfolded digestive tract tissue structure is in the same plane when rolled up, it can also be used to absorb excess neutral formalin solution in the digestive tract tissue structure.

[0023] Preferably, the specific method of using the above shaping device to unfold and roll up the whole digestive tract tissue structure that has been cut open on one side is: (1) First, install the first limiting plate on the winding rod, and then unfold the pharyngeal part of the digestive tract tissue structure and clamp it in the fixed groove of the winding rod; then install the second limiting plate on the winding rod; finally, install the organizer on the winding rod and cover the limiting plate, while allowing the digestive tract tissue structure to move out of the organizing groove.

[0024] (2) Carefully tighten the digestive tract tissue structure and rotate the winding rod so that the digestive tract tissue structure can be flattened under the action of the sorting groove, and the flattened digestive tract tissue structure is wound on the winding rod with the inner wall as the inner circle and the outer wall as the outer circle.

[0025] (3) After the winding is completed, the wound digestive tract tissue structure is tied and fixed from the outermost circle, and the shaping device is removed to obtain the shaped animal complete digestive tract tissue structure.

[0026] Preferably, in step (2), during the flattening and winding process, the finishing groove only contacts the outer wall of the digestive tract tissue structure, which can avoid damage to the villi surface of the inner wall of the digestive tract tissue structure caused by contact and friction, and is conducive to improving the integrity of the slices.

[0027] Among them, in step S3, the fixation treatment can prevent the autolysis and corruption of tissues caused by digestive juices, so that cells and tissues maintain their original morphology and structure, which is convenient for subsequent sectioning, staining and observation.

[0028] Preferably, the fixation treatment comprises: soaking the shaped animal whole digestive tract tissue structure in a neutral formalin solution for at least 24 hours to obtain a fixed whole digestive tract shaped structure.

[0029] Dehydration is an important prerequisite for ensuring transparency and wax immersion, and affects the quality of the slices. After fixation and rinsing, the digestive tract tissue will have a lot of residual water. Since water is insoluble in transparent reagents benzene and paraffin, in order to allow the transparent reagents and paraffin to fully penetrate the tissue, the residual water in the tissue needs to be removed before transparentization and wax immersion. If dehydration is not thorough, crypts will appear in the center of the embedded wax block, the transparency will be poor, and the slices will be cloudy.

[0030] Preferably, the dehydration treatment includes: soaking the fixed whole digestive tract tissue structure in gradient ethanol solutions with concentrations from low to high in sequence. The specific soaking time is reasonably controlled according to the tissue condition and experimental environment to ensure thorough dehydration to avoid affecting the subsequent immersion of transparent agent and paraffin.

[0031] The purpose of transparent treatment is to remove ethanol from the tissue and create conditions for paraffin to fully penetrate the tissue.

[0032] Preferably, the transparent treatment includes: soaking the dehydrated whole digestive tract tissue structure in xylene; the specific soaking time is reasonably controlled according to the tissue condition and experimental environment, so as to ensure that the ethanol is removed as thoroughly as possible.

[0033] The embedding treatment can make the digestive tract tissue structure have a certain hardness, which is convenient for later sectioning. Preferably, the embedding treatment includes: soaking and wrapping the whole digestive tract tissue structure after transparent treatment with molten paraffin. More preferably, the melting point of the paraffin is 56-62°C.

[0034] Preferably, the slicing process includes: firstly continuously slicing the wax block containing the entire digestive tract tissue structure to obtain a wax strip, then spreading the wax strip in constant temperature water, and finally fishing it out with a glass slide so that the wax strip is tightly attached to the glass slide, and obtaining the slice after water absorption and drying.

[0035] More preferably, the temperature of the constant temperature water is 45-50° C. The thickness of the slice is 2-5 μm; and the drying temperature is 63-67° C.

[0036] Tissues are stained to make different substances in the tissue cell structure appear in different colors for easy observation. The clarity of the staining affects the subsequent observation and diagnosis under a microscope.

[0037] Preferably, the staining process comprises: sequentially subjecting the dried slices to xylene dewaxing, gradient ethanol hydration, hematoxylin staining, water washing, hydrochloric acid ethanol differentiation, bluing, water washing, and eosin staining to obtain stained digestive tract tissue slices.

[0038] The purpose of sealing is to preserve the sections for later observation.

[0039] Preferably, the sealing process comprises: dripping neutral gum on the digestive tract tissue slices after the staining process, sealing after removing air with a cover glass, and obtaining continuous pathological slices of the entire digestive tract of the animal.

[0040] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. The method for preparing continuous pathological sections of the entire digestive tract of an animal of the present invention ensures that the fixative can fully penetrate into the inner and outer walls of the digestive tract and the mucosal fold structure by pre-fixing and shaping the tissue structure of the entire digestive tract of the animal, thereby effectively preventing the separation of the mucosal layer from the underlying tissue and greatly reducing the probability of tissue breakage and deformation.

[0041] 2. The method for preparing continuous pathological sections of the entire digestive tract of an animal of the present invention can effectively improve the curling problem of the esophagus, stomach and intestines during the dehydration and embedding process by pre-fixing and shaping the tissue structure of the entire digestive tract of the animal, thereby achieving high-precision flattening and shaping of the digestive tract.

[0042] 3. The method for preparing continuous pathological sections of the entire digestive tract of animals of the present invention has good universality, high stability, and strong operability, which significantly improves the development and shaping quality of the digestive tract tissue structure, ensures the continuity and integrity of the sections, avoids the shedding, deformation or dislocation of the tissue, and provides a full-view display for observing the expansion path and laws of digestive tract lesions, which is conducive to revealing the pathological changes of complex digestive tract diseases (such as digestive tract inflammation, tumors, etc.), and is suitable for large-scale promotion and application.

[0043] 4. The method for preparing continuous pathological sections of the entire digestive tract of an animal of the present invention reduces repeated operations and losses during the experimental process through one-time flattening and continuous slicing technology, and can quickly prepare high-quality continuous sections. Compared with traditional preparation methods, while ensuring the quality of the sections, it can shorten the preparation time by nearly 30%, greatly improving the production efficiency of continuous pathological sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the structure of the shaping device in Example 1 of the present invention.

[0045] Figure 2 It is a structural explosion diagram of the shaping device in Example 1 of the present invention.

[0046] Figure 3 It is a schematic diagram of the structure of the first limiting plate of the shaping device in Example 1 of the present invention.

[0047] Figure 4 It is a schematic diagram of the structure of the winding rod of the shaping device in Example 1 of the present invention (a is a front view, b is a top view).

[0048] Figure 5 It is a schematic diagram of the structure of the shaping device organizer in Example 1 of the present invention (a is a bottom view, b is a cross-sectional view along the AA plane).

[0049] Figure 6 This is an in vitro anatomical diagram of the entire digestive tract of a mouse in an embodiment of the present invention.

[0050] Figure 7 This is a photograph of the entire digestive tract tissue structure of mice after shaping in an embodiment of the present invention.

[0051] Figure 8 This is a photograph of the tissue structure of the entire digestive tract of mice during fixation in an embodiment of the present invention.

[0052] Fig. 9 This is a photo of the slice processing during slice production in an embodiment of the present invention.

[0053] Fig.10 This is a tissue slice prepared in an embodiment of the present invention.

[0054] Fig.11 It is a panoramic slice diagram of the digestive tract in an embodiment of the present invention.

[0055] Fig.12 This is the tissue slice produced in the comparative example of the present invention.

[0056] Fig.13 Esophageal section diagrams in the embodiments and comparative examples of the present invention (A is the embodiment, B is the comparative example).

[0057] Fig.14 Stomach section diagram in an embodiment of the present invention.

[0058] Fig.15 Duodenum section diagrams in the examples and comparative examples of the present invention (A is the example, B is the comparative example).

[0059] Fig.16 Jejunum sections in the examples and comparative examples of the present invention (A is the example, B is the comparative example).

[0060] Fig.17 Ileum section diagrams in the examples and comparative examples of the present invention (A is the example, B is the comparative example).

[0061] Fig.18 A cecal section diagram in an embodiment of the present invention.

[0062] Fig.19 This is a colon section diagram in an embodiment of the present invention.

[0063] Fig. 20 It is a cross-sectional view of the rectum and anus in an embodiment of the present invention.

[0064] Fig.21 It is a colorectal section diagram in the comparative example of the present invention.

[0065] Markings in the figure: 1-fixing groove; 2-organizer; 3-limiting protrusion; 4-winding rod; 5-first limiting plate; 6-organizing groove; 7-second limiting plate; 8-organizer hole; 9-limiting plate hole. DETAILED DESCRIPTION

[0066] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0067] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Example

[0068] A method for preparing continuous pathological sections of the entire digestive tract of a mouse comprises the following steps: S1: Dissect and obtain the entire digestive tract tissue structure of the animal.

[0069] Before dissecting and collecting the entire digestive tract tissue structure, the experimental mice were fasted for 12 hours to promote intestinal emptying; after the mice were killed, the digestive tract tissue structure was immediately dissected.

[0070] The specific dissection method is as follows: the mouse's glossopharynx, chest, and abdomen are cross-cut, the skin is pulled apart to completely expose the mouse's digestive tract, the pubic symphysis of the mouse's pelvis is cut open, and the rectum is repeatedly exposed; the mouse's pharynx is severed 3 mm away from the esophagus, and some throat tissue is retained; the mouse's trachea and esophagus are pulled together to the stomach, duodenum, jejunum, colon, cecum, rectum, and finally the anus to perform blunt separation of the intestinal tract and part of the mesenteric structure; after removing all the intestinal tissues, they are gently rinsed in 4°C saline to remove blood stains, intestinal contents, digestive fluid, etc. to prevent tissue damage.

[0071] S2: Pre-fix and shape the entire digestive tract tissue structure of the animal.

[0072] Pre-fixation treatment includes: carefully grasping the pharynx with forceps, and soaking the entire digestive tract tissue structure of the mouse in a neutral formalin solution (pH 7.3; mass concentration 10%) for 30 minutes; after pre-fixation for 30 minutes, it was found that the digestive tract tissue turned slightly white, and the blood vessels of the digestive wall were clearly visible ( Figure 6 ).

[0073] The steps of shaping treatment include: a. unilaterally cutting open the whole digestive tract tissue structure that has been pre-fixed along the digestive tract cavity, so that the whole digestive tract tissue structure can be unfolded along the cutting position.

[0074] b. The whole digestive tract tissue structure cut open on one side is unfolded and rolled into a concentric ring using a shaping device to obtain the whole digestive tract tissue structure of the animal after shaping ( Figure 7 ).

[0075] like Figures 1 to 5 As shown, the shaping device consists of a sorter, a winding rod and a limit plate; the sorter is rotatably mounted on the winding rod; the limit plate is located inside the sorter and is arranged through the winding rod; the sorter is provided with a sorting groove for flattening the entire digestive tract tissue structure; the winding rod is provided with a fixing groove; the limit plate includes a first limit plate and a second limit plate; the winding rod is also provided with a limit protrusion; the limit plate is a filter paper board.

[0076] The specific method of using a shaping device to unfold and roll the entire digestive tract tissue structure that has been cut open on one side is as follows: (1) first install the first limiting plate on the winding rod, and then unfold the pharyngeal part of the digestive tract tissue structure and clamp it in the fixed groove of the winding rod; then install the second limiting plate on the winding rod; finally, install the organizer on the winding rod and cover the limiting plate, while allowing the digestive tract tissue structure to move out of the organizing groove; (2) carefully tighten the digestive tract tissue structure and rotate the winding rod so that the digestive tract tissue structure can be flattened under the action of the organizing groove, and the flattened digestive tract tissue structure is wound on the winding rod with the inner wall as the inner circle and the outer wall as the outer circle; (3) after winding is completed, the wound digestive tract tissue structure is tied and fixed from the outermost circle, and the shaping device is removed to obtain the shaped animal entire digestive tract tissue structure.

[0077] S3: The shaped animal's entire digestive tract tissue structure is sequentially fixed, dehydrated, transparentized, embedded, sliced, stained and sealed to obtain continuous pathological sections of the animal's entire digestive tract.

[0078] Among them, fixation treatment: after removing the shaping device, put the shaped animal digestive tract tissue structure into an embedding box (such as Figure 8 As shown); Cover the embedding box and immerse the embedding box in neutral formalin at 40℃ for 2h (twice).

[0079] Dehydration treatment: The fixed animal digestive tract tissue structure was immersed in ethanol solutions of different concentrations in turn, and the temperature was the same as the fixed temperature of 40°C. The specific ethanol solution concentrations and immersion times were: 75% ethanol for 60 minutes, 85% ethanol for 50 minutes, 95% ethanol (first time) for 50 minutes, 95% ethanol (second time) for 50 minutes, 100% anhydrous ethanol (first time) for 40 minutes, and 100% anhydrous ethanol (second time) for 40 minutes.

[0080] Transparent treatment: The dehydrated digestive tract tissue structure was immersed in xylene at a fixed temperature of 40°C. The specific immersion time was: xylene (first time) 50 min, xylene (second time) 50 min, xylene (third time) 50 min.

[0081] Wax immersion treatment: The entire digestive tract tissue structure after transparency treatment is immersed in molten paraffin (melting point 54-56°C) at a temperature of 65°C. The specific time is: paraffin (first time) 50 minutes, paraffin (second time) 50 minutes, paraffin (third time) 50 minutes.

[0082] All processes from fixation to wax impregnation were performed in the fully automatic dehydration machine Leica PELORIS 3.

[0083] Embedding process: completed on Leica HistoCore Arcadia H embedding machine, embedding temperature 65 ℃.

[0084] Slice processing: Fig. 9 As shown, fix the slicer and the wax block on the slicer, adjust the inclination angle of the blade to 5°, adjust the scale pointer to 20μm, loosen the wheel holder, slowly push the wheel, and adjust the distance between the blade and the wax block; at the same time, hold the wax strip with a brush to observe the integrity of the wax strip; until the wax strip is complete, adjust the scale pointer to 3μm and continue slicing; select the complete ones from the cut wax strips, use a brush to gently hold them in a 48℃ constant temperature water bath for spreading, and after flattening, slowly extend the marked slide vertically into the water, approach the wax strip and slowly extend it in the vertical direction to fish out the slice, so that the wax strip is close to the slide; the picked up slide is placed flat on absorbent paper for 10 minutes, placed on a slide spreader, and dried at 65℃ for 10 minutes to obtain slices.

[0085] Staining treatment: the dried sections were subjected to xylene dewaxing, gradient ethanol hydration, hematoxylin staining, water washing, hydrochloric acid ethanol differentiation, bluing, water washing, eosin staining and other steps in sequence to obtain the digestive tract tissue sections after staining; the specific solutions and time were: xylene (first time) 5 min, xylene (second time) 5 min, 96% ethanol (first time) 1 min, 96% ethanol (first time) 2 min, 70% ethanol 2 min, tap water 2 min, hematoxylin staining solution 4 min, tap water 2 min, 1% hydrochloric acid ethanol 1 min, distilled water 3 min, bluing solution 2 min, demineralized water 2 min, 70% ethanol 30 s, 1% alcohol-soluble eosin staining solution 210 s, 96% ethanol 2 min, 99.9% ethanol (first time) 1 min, 99.9% ethanol (second time) 1 min, 99.9% ethanol 1 min (third time), and natural drying for 1 min.

[0086] Sealing treatment: Add neutral gum to the digestive tract tissue slices after staining, use a cover glass to remove air and seal, and obtain continuous pathological sections of the entire digestive tract of the animal (such as Fig.10 , Fig.11 as shown).

[0087] Staining and sealing are completed on Dako's HE staining and sealing machine (Dako CoverStainer).

[0088] Comparative Example A method for preparing continuous pathological sections of the entire digestive tract of a mouse comprises the following steps: S1: Dissect and obtain the entire digestive tract tissue structure of the animal.

[0089] Before dissecting and collecting the entire digestive tract tissue structure, the experimental mice were fasted for 12 hours to promote intestinal emptying; after the mice were killed, the digestive tract tissue structure was immediately dissected.

[0090] The specific dissection method is: cut the mouse's glossopharynx, chest, and abdomen in a cross shape, pull the skin apart, completely expose the mouse's digestive tract, cut the pubic symphysis of the mouse's pelvis, and repeatedly expose the rectum. Cut it off from the mouse's pharynx 3mm away from the esophagus, and retain some throat tissue. Pull the mouse's trachea and esophagus together to the stomach, duodenum, jejunum, colon, cecum, rectum, and even the anus to bluntly separate the intestinal tract and part of the mesenteric structure, and divide the digestive tract into 5 parts: esophagus, duodenum, jejunum, ileum, and colorectum (including colon, cecum, rectum, and anus). Then rinse them lightly with 4°C saline to remove blood stains, intestinal contents, digestive fluid, etc. to prevent tissue damage.

[0091] S2: The five parts of the digestive tract are fixed, dehydrated, transparentized, embedded, sliced, stained and sealed in sequence to obtain continuous pathological sections of the five parts of the animal's digestive tract.

[0092] Among them, fixation treatment: after removing the shaping device, put the shaped animal digestive tract tissue structure into the embedding box. Cover the embedding box, put the embedding box into 40℃ neutral formalin and soak it for 2 hours, repeat the soaking twice.

[0093] Dehydration treatment: The fixed animal digestive tract tissue structure was immersed in ethanol solutions of different concentrations in turn, and the temperature was the same as the fixed temperature of 40°C. The specific ethanol solution concentrations and immersion times were: 75% ethanol for 60 minutes, 85% ethanol for 50 minutes, 95% ethanol (first time) for 50 minutes, 95% ethanol (second time) for 50 minutes, 100% anhydrous ethanol (first time) for 40 minutes, and 100% anhydrous ethanol (second time) for 40 minutes.

[0094] Transparent treatment: The dehydrated digestive tract tissue structure was immersed in xylene at a fixed temperature of 40°C. The specific immersion time was: xylene (first time) 50 min, xylene (second time) 50 min, xylene (third time) 50 min.

[0095] Wax immersion treatment: The entire digestive tract tissue structure after transparency treatment is immersed in molten paraffin (melting point 54-56°C) at a temperature of 65°C. The specific time is: paraffin (first time) 50 minutes, paraffin (second time) 50 minutes, paraffin (third time) 50 minutes.

[0096] All processes from fixation to wax impregnation were performed in the fully automatic dehydration machine Leica PELORIS 3.

[0097] Embedding process: completed on Leica HistoCore Arcadia H embedding machine, embedding temperature 65 ℃.

[0098] Slicing: Fix the slicer and wax block on the slicer, adjust the blade angle to 5°, adjust the scale pointer to 20μm, loosen the wheel holder, slowly push the wheel, and adjust the distance between the blade and the wax block. At the same time, hold the wax tape with a brush to observe the integrity of the wax tape. Until the wax tape is complete, adjust the scale pointer to 3μm and continue slicing. Select the complete ones from the cut wax tapes, gently hold them with a brush and put them into a 48℃ constant temperature water tank for spreading. After flattening, slowly extend the marked slide vertically into the water, approach the wax tape and slowly extend it in the vertical direction to fish out the slice, so that the wax tape is close to the slide. Lay the picked up slide flat on absorbent paper for 10 minutes, place it on the spreader, and dry it at 65℃ for 10 minutes to obtain the slice.

[0099] Staining treatment: The dried sections were sequentially subjected to xylene dewaxing, gradient ethanol hydration, hematoxylin staining, water washing, hydrochloric acid ethanol differentiation, blueing, water washing, eosin staining and other steps to obtain the digestive tract tissue sections after staining. The specific solution and time are: xylene (first time) 5min, xylene (second time) 5min, 96% ethanol (first time) 1min, 96% ethanol (first time) 2min, 70% ethanol 2min, tap water 2min, hematoxylin staining solution 4min, tap water 2min, 1% hydrochloric acid ethanol 1min, distilled water 3min, blueing solution 2min, demineralized water 2min, 70% ethanol 30s, 1% alcohol-soluble eosin staining solution 210s, 96% ethanol 2min, 99.9% ethanol (first time) 1min, 99.9% ethanol (second time) 1min, 99.9% ethanol 1min (third time), and natural drying for 1min.

[0100] Sealing treatment: Add neutral gum to the digestive tract tissue slices after staining, use a cover glass to remove air and seal, and obtain animal digestive tract pathological slices (such as Fig.12 as shown).

[0101] Staining and sealing are completed on Dako's HE staining and sealing machine (Dako CoverStainer).

[0102] Experimental example: Observe the pathological sections obtained in the embodiment and the comparative example respectively, Wherein, embodiment esophageal section is as follows Fig.13 A (where 1 is the esophageal section and 2 is the section at the junction of the esophagus and stomach). Fig.13B (where 1 is a panoramic slice of the esophagus, and 2 is a local enlarged slice of the esophagus).

[0103] Example Stomach slices Fig.14 (Among them, 1 is a slice of the stomach, and 2 is a slice of the junction of the stomach and duodenum).

[0104] Example Duodenum slices Fig.15 A (where 1 is a section of the duodenum, and 2 is a section of the duodenum and jejunum). Fig.15 As shown in B (where 1 is a panoramic slice of the duodenum, and 2 is a partial enlarged slice of the duodenum).

[0105] Example jejunum slices Fig.16 A (where 1 is a jejunal section, and 2 is a section at the junction of the jejunum and ileum). Fig.16 As shown in B (where 1 is a panoramic slice of the jejunum, and 2 is a partial enlarged view of the jejunum slice).

[0106] Example Ileum slices Fig.17 As shown in Figure A (where 1 is a slice of the ileum and 2 is a slice of the junction of the ileum and cecum). Fig.17 As shown in B (where 1 is a panoramic slice of the ileum, and 2 is a partial enlarged slice of the ileum).

[0107] Example Cecum slices Fig.18 (Among them, 1 is a slice of the cecum, and 2 is a slice of the junction of the cecum and colon).

[0108] Example Colon section Fig.19 (Among them, 1 is a section of the colon, and 2 is a section of the junction of the colon and rectum).

[0109] Example Rectal and anal sections as Fig. 20 As shown (where 1 is a rectal section, 2 is a section at the junction of the rectum and anus, and 3 is a section of the anus).

[0110] Comparative colorectal sections Fig.21 (Among them, 1 is a panoramic section of the colorectum, and 2 is a local enlarged section of the colorectum).

[0111] According to the observation results of the slices in the embodiments and the comparative examples, it can be known that the mouse digestive tract slices prepared by the method of the present invention, especially by using a shaping device with a simple structure, easy operation, and rapid unfolding and winding of the digestive tract tissue structure, ensure that the fixative can fully penetrate the inner and outer walls of the digestive tract and the mucosal fold structure, prevent the separation of the mucosal layer from the underlying tissue, greatly reduce tissue breakage and deformation, and prevent the esophagus, stomach and intestines from curling during dehydration and embedding, thereby achieving high-precision flattening and shaping of the digestive tract; therefore, compared with the slices prepared by the existing methods, the slices obtained by the preparation method of the present invention have the advantages of less shedding, deformation or dislocation of digestive tract tissue, clear slice texture, and good continuity and integrity of the slices, providing a full-view display for observing the expansion path and laws of digestive tract lesions, which is conducive to revealing the pathological changes of complex digestive tract diseases and is suitable for large-scale promotion and application.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing continuous pathological sections of the entire digestive tract of an animal, characterized in that: The following steps are involved: S1: Obtain the entire digestive tract tissue of the animal by dissection; S2: Pre-fix and shape the entire digestive tract tissue of the animal; The pre-fixation treatment includes: soaking the entire digestive tract tissue structure of the animal in a neutral formalin solution for 25-35 minutes; The shaping process comprises: a. The whole digestive tract tissue that has been pre-fixed is unilaterally cut open along the digestive tract cavity, so that the whole digestive tract tissue structure can be unfolded along the cut position; b. unfolding the whole digestive tract tissue structure cut open on one side and rolling it into a concentric ring to obtain the shaped whole digestive tract tissue of the animal; S3: The shaped animal whole digestive tract tissue is sequentially fixed, dehydrated, transparentized, embedded, sliced, stained and sealed to obtain continuous pathological sections of the animal whole digestive tract.

2. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 1, characterized in that: In step S1, the whole digestive tract tissue includes the complete and continuous esophagus, stomach, duodenum, jejunum, colon, cecum, rectum and anus of the animal.

3. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 1, characterized in that: In step b, when winding into concentric rings, the inner wall of the expanded digestive tract tissue structure is used as the inner circle and the outer wall is used as the outer circle, and the expanded digestive tract tissue structure is ensured to be in the same plane when winding.

4. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 1, characterized in that: In step b, a shaping device is used to perform winding and shaping; The shaping device comprises a finisher, a winding rod and a limiting plate; The organizer is rotatably mounted on the winding rod; the limit plate is located inside the organizer and is arranged through the winding rod; the organizer is provided with an organizing groove for flattening the entire digestive tract tissue structure; The winding rod is provided with a fixing groove for clamping and fixing one end of the whole digestive tract tissue structure; The limiting plate includes a first limiting plate and a second limiting plate, which are used to ensure that the unfolded digestive tract tissue structure is in the same plane when rolled.

5. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 4, characterized in that: The winding rod is also provided with a limiting protrusion for preventing the first limiting plate from sliding downward along the winding rod.

6. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 4, characterized in that: The specific method of using the shaping device to unfold and roll the entire digestive tract tissue structure that has been cut open on one side is as follows: (1) First, install the first limiting plate on the winding rod, then unfold the pharyngeal part of the digestive tract tissue structure and clamp it in the fixing groove of the winding rod; then install the second limiting plate on the winding rod; Finally, the organizer is installed on the winding rod and covered with a limiting plate, and the digestive tract tissue structure is moved out of the organizing tank; (2) Carefully tighten the digestive tract tissue structure and rotate the winding rod so that the digestive tract tissue structure can be flattened under the action of the sorting groove, and the flattened digestive tract tissue structure is wound on the winding rod with the inner wall as the inner circle and the outer wall as the outer circle; (3) After the winding is completed, the wound digestive tract tissue structure is tied and fixed from the outermost circle, and the shaping device is removed to obtain the shaped animal complete digestive tract tissue structure.

7. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 1, characterized in that: In step S3, the fixation treatment includes: soaking the shaped animal digestive tract tissue structure in a neutral formalin solution for at least 24 hours.

8. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 1, characterized in that: In step S3, the dehydration treatment includes: soaking the whole digestive tract tissue structure after fixation in gradient ethanol solutions with concentrations from low to high in sequence.

9. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 1, characterized in that: In step S3, the slicing process includes: firstly continuously slicing the wax block containing the entire digestive tract tissue structure to obtain a wax strip, then spreading the wax strip in constant temperature water, and finally fishing it out with a glass slide so that the wax strip is tightly attached to the glass slide, and obtaining a slice after water absorption and drying.

10. The method for preparing continuous pathological sections of the entire digestive tract of an animal according to claim 1, characterized in that: In step S3, the staining process includes: sequentially performing xylene dewaxing, gradient ethanol hydration, hematoxylin staining, water washing, hydrochloric acid ethanol differentiation, blueing, water washing, and eosin staining on the dried slices to obtain digestive tract tissue slices after staining.

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