Paraffin embedding and section preparation method of mesenteric tissue of stichopus japonicus

Abstract

The invention discloses a paraffin embedding and slice preparation method for mesenteric tissue of stichopus japonicus, and belongs to the technical field of biological histology. The method aims at solving the problems that the tissue is ultrathin and soft, and is easy to deform and adhere, so that a slice structure is damaged, and a microscopic imaging structure is not clear. According to three core improvements, in-situ pre-fixation of living bodies is combined with oscillation stretching (shearing sampling and oscillation anti-adhesion after bohn liquid is dropwise added), it is guaranteed that mesentery is not separated from liquid in the whole process (anti-adhesion), dyeing auxiliary positioning is carried out after transparentizing (eosin dyeing improves the visibility of transparent tissue), and shaking table dynamic treatment is carried out in the whole process (the stages of fixation, washing and dehydration); and the mechanical damage risk is obviously reduced. According to the method, the integrity of the original form of mesentery is maintained, meanwhile, the problems of tissue adhesion, fracture, slice cavity and incapability of distinguishing the form are solved, the continuous and complete 5-8 mu m slices are successfully prepared, the tissue structure definition is remarkably improved, and the tissue quality is improved. And a reliable technical basis is provided for high-resolution histological analysis of mechanisms such as intestinal regeneration and immune response of the stichopus japonicus.

Description

Technical Field

[0001] The invention relates to a paraffin embedding and section preparation method for sea cucumber mesenteric tissue, which is suitable for subsequent microscopic analysis such as histopathological observation, immunohistochemistry, in situ hybridization, etc., and belongs to the technical field of biological histology. Background Art

[0002] Sea cucumber ( Apostichopus japonicus Selenka (Selena) belongs to the phylum Echinodermata, class Acipenseridae, order Scutochoris. Due to its remarkable intestinal regenerative capacity, sea cucumbers have become a model for regeneration research. The mesentery, as the epicenter of intestinal regeneration, has become crucial for this study. However, due to the unique characteristics of the sea cucumber mesenteric tissue, current paraffin embedding methods have significant limitations for directly observing the fine structure of the mesenteric tissue. The mesentery is tiny and extremely thin (appearing as a translucent membrane). Its inherent rigidity and exceptionally soft texture make it prone to curling, folding, and self-adhesion during sample handling. It is also extremely sensitive to mechanical manipulations (such as transfer, dehydration, clearing, and wax immersion), and its structural integrity is easily compromised. Conventional paraffin embedding procedures can easily cause tissue deformation, excessive shrinkage, and even tearing. This is reflected in sections as severe tissue incompleteness (fragmentation and cracking), internal cavities (collapse of areas due to insufficient wax immersion), and distorted tissue stacking, leading to blurred microstructures. This severely hinders subsequent precise observation and analysis.

[0003] Paraffin embedding is a crucial technique in histological research. Fixing, dehydrating, and transparent soft biological tissues is then infiltrated and solidified with paraffin, enabling ultrathin (typically micrometer-thick) serial sectioning. High-quality sections are a prerequisite for precise microscopic morphological observations (such as H&E staining), specific molecular localization (such as immunohistochemistry and in situ hybridization), and other histological analysis methods, directly determining the clarity, accuracy, and reliability of the observations.

[0004] Conventional procedures for embedding and transparentizing the sea cucumber mesentery present a series of challenges, posing a significant challenge to research related to the sea cucumber intestine and its regeneration. Therefore, the present invention provides a paraffin-embedded sample preparation method specifically adapted for this tissue. This method has significant practical value and scientific research significance for successfully obtaining structurally intact and well-defined sea cucumber mesentery sections, enabling high-resolution histological analysis of key physiological and pathological processes such as digestion, immunity, and regeneration. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies in the existing technology and provide a method for paraffin embedding and sectioning of sea cucumber mesenteric tissue. Through an original operating process, the problems of section structure destruction and section voids caused by the ultra-thin and fragile tissue are solved, and sea cucumber mesenteric slices with complete structure and clear layers are successfully prepared.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention first provides a paraffin embedding and sectioning method for sea cucumber mesenteric tissue, comprising the following steps: (1) In situ pre-fixation to prevent deformation and sampling: The anesthetized dissected sea cucumber is fixed on the dissection board, and Bonn's solution is dripped on the surface of the target mesentery to achieve in situ pre-fixation; pinch one end of the mesentery with forceps and cut a rectangular piece of tissue, and transfer the rectangular tissue to a centrifuge tube containing Bonn's solution by dragging it with the pinched end, shake it to stretch it, and then fix it on a shaker; (2) Dynamic rinsing: After the rectangular tissue in step (1) is fixed, the rectangular tissue is rinsed with phosphate buffered saline (PBS) under shaking conditions to completely remove the residual Bonn's solution; (3) Gradient dehydration: The rectangular tissue after dynamic rinsing in step (2) was dehydrated in a shaker with 70%, 80%, 90%, 95%, 100% I, and 100% II ethanol in a gradient manner; (4) Transparent treatment: the rectangular tissue after gradient dehydration in step (3) is transferred into transparent agent I and transparent agent II for graded transparentization; (5) Staining-assisted positioning: The rectangular tissue that has been transparentized in step (4) is immersed in eosin staining solution for staining. After staining, it is rinsed with phosphate buffered saline (PBS) to significantly improve the recognition of the embedding direction; (6) Paraffin impregnation and embedding: The rectangular tissue stained in step (5) is sequentially transferred into molten paraffin I and paraffin II for immersion under normal pressure; (7) Embedding and sectioning: Place the rectangular tissue soaked in wax in step (6) in an embedding mold, pour molten paraffin, cool and solidify at 4°C, and then slice continuously with a microtome to obtain sea cucumber mesenteric tissue sections, which are sea cucumber mesenteric paraffin-embedded samples.

[0007] In the above technical solution, in step (1), the amount of Bonn's solution added is 40-60 μL, and after addition, the solution is allowed to stand for 1 minute to achieve in situ pre-fixation.

[0008] In the above technical solution, in step (1), a rectangular section of mesentery with a length of 1.5-1.8 cm is cut, and the mesentery is kept from being separated from the liquid when dragging and transferring the rectangular tissue to prevent adhesion, and the rectangular tissue is transferred to a centrifuge tube containing 5 mL of Bonn's solution.

[0009] In the above technical solution, in step (1), the rectangular tissue is transferred to a centrifuge tube containing Bonn's solution and then tapped on the operating table three times; then, the tissue is shaken and stretched to prevent adhesion, and the shaking and stretching is performed by gently shaking the centrifuge tube in a horizontal direction 1-3 times with an oscillation amplitude of 5-10 cm; finally, it is placed on a side-swing shaker at room temperature for 20-28 hours, preferably for 24 hours, and the shaking frequency of the shaker is 15-25 times / minute.

[0010] In the above technical solution, in step (2), the phosphate buffer solution is rinsed 2-4 times, each time for 10-30 minutes; the number of rinses is preferably 3 times, and each rinse time is preferably 10 minutes; the shaking table condition is a swing frequency of 15-25 times / minute.

[0011] In the above technical solution, in step (3), the volume of each concentration of ethanol is 50 times the volume of the rectangular tissue, and the dehydration time of each concentration of ethanol is independently 1.5-2.5 hours; the shaking frequency of each concentration of ethanol is 15-25 times / minute.

[0012] In the above technical solution, in step (4), the types of the transparent agent I and the transparent agent II can be the same or different, and can be any one of the environmentally friendly transparent agents of xylene or limonene; the dosage of the transparent agent I and the transparent agent II is the same, and their volume is ≥10 times that of the rectangular tissue; the graded transparentization has a transparent time of 25-35 minutes for each level.

[0013] In the above technical solution, in step (5), the eosin staining is performed for 10-20 seconds; and the phosphate buffered saline (PBS) rinsing is performed for 10-30 seconds.

[0014] In the above technical solution, in step (6), the molten paraffin has a melting point of 56-60°C; the immersion is carried out twice, once in paraffin I and once in paraffin II, and each immersion time is 1-2 hours.

[0015] In the above technical solution, in step (7), the embedding mold is preheated to 60°C before the wax-soaked rectangular tissue is placed therein; the continuous sectioning has a thickness of 5-8 μm.

[0016] In the above technical solution, the shaking of steps (1), (2) and (3) are all carried out on a side-swing shaking table with a swing frequency of 15-25 times / minute, thereby reducing the risk of mechanical damage throughout the process; the staining positioning operation in step 5 breaks through the traditional process sequence and effectively avoids the distortion of the tissue structure caused by embedding misalignment.

[0017] The present invention also provides a sea cucumber mesenteric tissue slice obtained by the above preparation, with a slice thickness of 5-8 μm and no stacking, distortion or tissue rupture.

[0018] Compared with the existing technology, it has the following characteristics: This method addresses the issues of thin, soft, and easily deformable adhesions of sea cucumber mesentery, which can lead to structural damage in sections and unclear microscopic imaging. By combining in situ pre-fixation with oscillatory stretching (shear sampling after Bonn's solution is added, and oscillation prevents adhesions), ensuring that the tissue remains attached to the liquid throughout the process (to prevent adhesions), post-transparency staining to aid positioning (eosin staining enhances the visibility of transparent tissue), and dynamic shaking throughout the entire process (fixation, washing, and dehydration). This method significantly reduces the risk of mechanical damage. While maintaining the original morphological integrity of the mesentery, it also addresses the issues of tissue rupture and section voids. The method successfully produces continuous, complete 5-8 μm sections, significantly improving tissue structural clarity. This method provides a reliable technical foundation for high-resolution histological analysis of mechanisms such as intestinal regeneration and immune responses in sea cucumbers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HE image of a paraffin section sample of sea cucumber mesenteric tissue prepared using the traditional paraffin embedding process, where: Figure 1-1 HE image at 100 µm. Figure 1-2 HE image at 20 µm ( Figure 1-2 a is the adhesion and curled mesentery. Figure 1-2 b is a fragment of ruptured mesentery); Figure 2 This is a HE image of a paraffin section sample of the sea cucumber mesentery tissue obtained in Example 1 of the present invention, wherein: Figure 2-1 HE image at 100 µm. Figure 2-2 HE image at 20 µm ( Figure 2-2 a is the stretched mesenteric tip, Figure 2-2 b: The middle part of the mesentery is stretched). DETAILED DESCRIPTION

[0020] The following describes in detail the specific implementation of the technical solution of the present invention, but the present invention is not limited to the following description: The materials and reagents used in the embodiments of the present invention are as follows: 1. Materials Sample source: Live sea cucumber ( Apostichopus japonicus ) were purchased from Dalian Taiping Aquaculture Company, weighing 110±5g.

[0021] 2. Key reagents: The technical solution of the present invention is described in detail below with reference to specific embodiments: Example 1:

[0022] A method for preparing a paraffin-embedded sample of sea cucumber mesentery comprises the following steps: (1) In-situ pre-fixation to prevent deformation and sampling of living bodies: Place the anesthetized sea cucumber ventrally upward and fix the arms and legs to the dissecting board with insect pins (avoid pulling the mesentery); Use a pipette to precisely drop 50 μL of Bouin's solution onto the surface of the target mesentery (covering ≥80%) and let it sit for 1 minute; Use pointed forceps to pinch a 1 mm wide area of ​​the free end of the mesentery, and use dissecting scissors to cut a 1.5 cm long rectangular tissue along the edge of the drip area; Use the pinching end to drag the rectangular tissue into a 5 mL centrifuge tube containing 5 mL of Bouin's solution. Keep the mesentery in the liquid phase to prevent adhesion during the dragging transfer. Tap the tube wall lightly on the table three times and shake it horizontally twice (amplitude 5-10 cm / time). Place on a side-to-side rocking shaker (IKA, KS260) at 25°C for 24 h at a frequency of 20 beats / min; (2) Dynamic rinsing: Transfer the fixed rectangular tissue into a 50 mL centrifuge tube, add 40 mL PBS and rinse on a shaker (20 times / min), changing the medium every 10 minutes, and repeat 3 times (total time 30 minutes); (3) Gradient dehydration: The washed rectangular tissue was subjected to gradient dehydration in a 50 mL centrifuge tube. The volume of ethanol at each level was 50 times the volume of the tissue (≈30 mL). The shaking conditions were the same as those for rinsing, and the dehydration time for each level was 1 hour. (4) Transparent processing: Transfer the dehydrated tissue into a glass weighing dish and immerse in xylene (volume ≥ 10 times that of the tissue): xylene I: 30 min (standing), xylene II: 30 min (standing); (5) Staining-assisted positioning: After the transparent treatment, the rectangular tissue was picked up by the edge with blunt forceps and transferred to the eosin staining solution, immersed for 10 seconds, and immediately transferred to the PBS rinse solution and gently shaken for 10 seconds (until no red floating color is precipitated); (6) Paraffin impregnation: The stained rectangular tissue was transferred into molten paraffin (Leica Histowax, constant temperature in a 58°C water bath): Paraffin I: immersion for 60 minutes (standing), Paraffin II: immersion for 60 minutes (standing); (7) Embedding and sectioning: The embedding mold was preheated to 60°C, and the long axis of the tissue was placed parallel to the bottom of the mold. Molten paraffin was poured and solidified on a 4°C cold plate. After the wax block solidified, it was serially sectioned using a microtome (Leica RM2235) with a thickness of 6 μm.

[0023] Effect verification:

[0024] The tissue section samples of the mesentery obtained by the traditional paraffin embedding process and the paraffin embedding sample preparation method in Example 1 of the present invention were subjected to hematoxylin-eosin (HE) staining for comparison: Traditional paraffin embedding process: (1) Sampling: Fresh tissue was fixed in 4% paraformaldehyde for more than 24 hours. The tissue was removed from the fixative and trimmed with a scalpel in a fume hood. The trimmed tissue and the corresponding label were placed in a dehydration box.

[0025] (2) Dehydration: Place the dehydration box in the hanging basket and dehydrate in the dehydrator in a gradient of alcohol. 75% alcohol for 4 hours - 85% alcohol for 2 hours - 90% alcohol for 2 hours - 95% alcohol for 1 hour - anhydrous ethanol I for 30 minutes - anhydrous ethanol II for 30 minutes - benzene for 5-10 minutes - xylene I for 5-10 minutes - xylene II for 5-10 minutes - wax I for 1 hour - wax II for 1 hour - wax III for 1 hour.

[0026] (3) Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax into the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, remove the wax block from the embedding frame and trim the wax block.

[0027] (4) Sectioning: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4 μm. Float the slices on a 40°C warm water slide to flatten the tissue. Pick up the tissue with a glass slide and bake it in a 60°C oven. After the water is dried and the wax is melted, remove the slices and store them at room temperature for later use.

[0028] HE images of paraffin sections of sea cucumber mesenteric tissue prepared by traditional paraffin embedding process Figure 1 ( Figure 1-1 and Figure 1-2 ), the HE image of the paraffin section sample of the sea cucumber mesentery tissue obtained in Example 1 of the present invention is as shown in Figure 2 ( Figure 2-1 and Figure 2-2 ), as shown by Figure 1 and Figure 2It can be seen that the traditional paraffin embedding in the preparation of paraffin sections of sea cucumber mesentery inevitably causes adhesion and damage of the mesentery, which makes the tissue morphology after HE staining difficult to distinguish and rupture ( Figure 1-1 and Figure 1-2 ), which greatly limits the observation of the morphology and cells of the mesentery of sea cucumbers. The present invention uses four technical innovations: in situ fixation of living body, transfer of tissue in liquid and anti-adhesion by vibration, dynamic processing and staining positioning. The prepared paraffin sections can clearly observe the tissue morphology and cell layer of the mesentery ( Figure 2-1 and Figure 2-2 ), greatly improved the integrity of the sea cucumber mesentery, eliminated adhesion, stacking and rupture defects between tissues, and provided high-quality histological samples for the study of the intestinal regeneration mechanism of sea cucumbers.

[0029] The above examples are only for illustrating the technical concept and technical features of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent transformation or modification made based on the essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for paraffin embedding and sectioning of sea cucumber mesenteric tissue, characterized in that: The following steps are involved: (1) In situ pre-fixation to prevent deformation and sampling: The anesthetized dissected sea cucumber is fixed on the dissection board, and Bonn's solution is dripped on the surface of the target mesentery to achieve in situ pre-fixation; pinch one end of the mesentery with forceps and cut a rectangular piece of tissue, and transfer the rectangular tissue to a centrifuge tube containing Bonn's solution by dragging it with the pinched end, shake it to stretch it, and then fix it on a shaker; (2) Dynamic rinsing: After the rectangular tissue in step (1) is fixed, the rectangular tissue is rinsed with phosphate buffered saline (PBS) under shaking conditions to completely remove the residual Bonn's solution; (3) Gradient dehydration: The rectangular tissue after dynamic rinsing in step (2) was dehydrated in a gradient manner using 70%, 80%, 90%, 95%, 100% I, and 100% II ethanol on a shaking table; (4) Transparent treatment: the rectangular tissue after gradient dehydration in step (3) is transferred into transparent agent I and transparent agent II for graded transparentization; (5) Staining-assisted positioning: The rectangular tissue that has been transparentized in step (4) is immersed in eosin staining solution for staining. After staining, it is rinsed with phosphate buffered saline (PBS) to significantly improve the recognition of the embedding direction; (6) Paraffin impregnation and embedding: The rectangular tissue stained in step (5) is sequentially transferred into molten paraffin I and paraffin II for immersion under normal pressure; (7) Embedding and sectioning: Place the rectangular tissue soaked in wax in step (6) in an embedding mold, pour molten paraffin, cool and solidify at 4°C, and then slice continuously using a microtome to obtain sea cucumber mesenteric tissue sections, which are sea cucumber mesenteric paraffin-embedded samples.

2. The preparation method according to claim 1, characterized in that In step (1), the amount of Bonn's solution added is 40-60 μL, and after addition, it is allowed to stand for 1 minute to achieve in situ pre-fixation; a rectangular section of mesentery with a length of 1.5-1.8 cm is cut, and when dragging the rectangular tissue to transfer it, the mesentery is kept from separating from the liquid to prevent adhesion, and the rectangular tissue is transferred to a centrifuge tube containing 5 mL of Bonn's solution.

3. The preparation method according to claim 1, characterized in that In step (1), the rectangular tissue is transferred to a centrifuge tube containing Bonn's solution and tapped on the operating table three times; then, the tissue is shaken and stretched to prevent adhesion, and the shaking and stretching is performed by gently shaking the centrifuge tube in a horizontal direction 1-3 times with an amplitude of 5-10 cm; finally, the tissue is placed on a side-swing shaker at room temperature for 20-28 hours, and the shaking frequency of the shaker is 15-25 times / minute.

4. The preparation method according to claim 1, characterized in that In step (2), the phosphate buffer solution is rinsed 2-4 times, each time for 10-30 minutes; and the shaking condition is a swing frequency of 15-25 times / minute.

5. The preparation method according to claim 1, characterized in that In step (3), the volume of each concentration of ethanol is 50 times the volume of the cuboid tissue, and the dehydration time of each concentration of ethanol is independently 1.5-2.5 hours; the shaking frequency of each concentration of ethanol is 15-25 times / minute.

6. The preparation method according to claim 1, characterized in that In step (4), the types of the transparent agent I and the transparent agent II can be the same or different, and can be any one of the environmentally friendly transparent agents of xylene or limonene; the dosage of the transparent agent I and the transparent agent II is the same, and their volume is ≥ 10 times that of the rectangular tissue; the graded transparentization has a transparent time of 25-35 minutes for each level.

7. The preparation method according to claim 1, characterized in that In step (5), the eosin staining is performed for 10-20 seconds; and the phosphate buffered saline (PBS) rinsing is performed for 10-30 seconds.

8. The preparation method according to claim 1, characterized in that In step (6), the melting point of the molten paraffin is 56-60°C; the immersion is carried out twice, once in paraffin I and once in paraffin II, and each immersion time is 1-2 hours.

9. The preparation method according to claim 1, characterized in that In step (7), the embedding mold is preheated to 60°C and then the wax-soaked rectangular tissue is placed therein; the continuous sectioning has a thickness of 5-8 μm.

10. A sea cucumber mesenteric tissue slice prepared by the preparation method according to any one of claims 1 to 9, characterized in that: The slice thickness was 5-8 μm, without stacking distortion or tissue breakage.

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