A method for preparing mouse intestinal tissue wax block for slicing

Abstract

The present invention provides a method for preparing a mouse intestinal tissue wax block for slicing, which comprises the following steps: sampling of intestinal tissue, fixation of intestinal tissue, dehydration of intestinal tissue, and embedding of intestinal tissue. The present invention has simple steps and convenient operation. The embedded intestinal tissue wax block can keep the intestinal tissue morphology intact during subsequent slicing, without or only occasionally showing muscle layer separation phenomenon; the connective tissue morphology of the submucosal layer is completely presented, which is convenient for scientific researchers to conduct comprehensive observation of specimens, and provides reliable materials for theoretical research on intestinal diseases.

Description

Technical field:

[0002] The invention relates to the technical field of pathological examination, in particular to a method for preparing a wax block of mouse intestinal tissue for slicing. Background technology:

[0004] The mouse intestinal disease model simulates human intestinal diseases in vivo under the action of artificial pathogenic factors (physical, chemical and biological factors), providing a theoretical basis for the prevention and treatment of diseases. On the basis of a reliable model, the corresponding histological sections are of great significance for detecting and observing the pathological changes of intestinal tissue. In terms of histology, the intestine can be divided into the mucosal layer, the muscularis mucosa, the submucosal layer, the muscular layer and the serosa. The submucosal layer is rich in blood vessels, lymphatic vessels and nerve networks, and these tissue structures are also emerging hotspots for the study of intestinal diseases. Therefore, the microscopic imaging of intact intestinal tissue is crucial for observing the occurrence and development of intestinal diseases.

[0005] However, current mouse intestinal slicing techniques generally have Figure 1 As shown in the figure, there is a gap Y between the muscular layer Z and the mucosal layer X, and the muscular layer of the mucosa is defective, which is not only not conducive to the overall histological analysis of the specimen intestinal tract, but also hinders the further discovery of unknown pathological changes that may exist in the submucosal layer. Summary of the invention:

[0007] The present invention aims to overcome the deficiencies in the prior art and provides a method for preparing a mouse intestinal tissue wax block for slicing.

[0008] This application provides the following technical solutions:

[0009] A method for preparing a wax block of mouse intestinal tissue for slicing, comprising the following steps: step A: sampling of intestinal tissue, characterized in that: step B: fixing of intestinal tissue, step C: dehydration of intestinal tissue, and step D: embedding of intestinal tissue;

[0010] Step B includes:

[0011] (1) Take out the embedding box, and pre-lay a first filter paper layer on the bottom of the embedding box; lay at least two first non-woven fabric layers of the same size on the first filter paper layer, cut the intestinal tract in the middle, and transfer the intestinal tissue to the middle position of the non-woven fabric; flatten the intestinal tissue, spread it into a rectangle, and fix it;

[0012] (2) Cover the flattened intestinal tissue with at least two layers of the second non-woven fabric layer of the same size, and then cover the second filter paper layer on the second non-woven fabric layer, and then press the filter paper with the index finger to flatten the intestinal tissue, thereby forming an intestinal tissue sandwich;

[0013] (3) Stapling the intestinal interlayer around; placing the stapled intestinal interlayer into a tissue embedding box; covering the intestinal interlayer with an anti-pressure layer, and then covering the embedding box. The anti-pressure layer is deformed in the squeezing direction of the embedding box cover to press the intestinal tissue interlayer below to prevent it from moving in the embedding box;

[0014] (4) Rinse the embedding cassette with running water for 17-25 minutes;

[0015] Step C includes placing the embedding cassette into a tissue dehydrator for dehydration;

[0016] Step D includes injecting wax water into the embedding mold, then opening the embedding box to take out the dehydrated intestinal tissue layer, opening the non-woven fabric to take out the intestinal tissue, placing the intestinal tissue in the wax water and repeatedly rinsing until bubbles are obvious on the surface, placing the longitudinal surface of the intestinal tissue on the embedding mold so that it is perpendicular to the bottom surface of the embedding mold, moving the mold to a freezing table, and when the wax water at the bottom of the mold solidifies, continuing to add wax water to the mold until the mold is full, and then placing the embedding box base upside down on the wax block that is about to solidify, and after the wax block is completely solidified, holding the embedding box base to take the wax block out of the embedding mold to obtain a wax block for slicing.

[0017] On the basis of the above technical solutions, there are also the following further technical solutions:

[0018] In step B, the first filter paper layer is covered with three layers of non-woven fabrics, and the intestinal tissue is covered with three layers of non-woven fabrics.

[0019] In step B, a stapler is used to staple the intestinal tissue layer in the thickness direction, and the stapler position needs to be 6-10 mm away from the edge of the tissue.

[0020] In the cylinder group of the tissue dehydrator in step C, ethanol is placed in cylinders 1 to 7, xylene is placed in cylinders 8 and 9, and slice paraffin is placed in cylinders 10 to 12.

[0021] The ethanol concentration in the first cylinder is 50%, the ethanol concentration in the second cylinder is 70%, the ethanol concentration in the third cylinder is 80%, the ethanol concentration in the fourth cylinder is 95%, and the ethanol concentrations in the fifth to seventh cylinders are 100%.

[0022] The dehydration time of the embedding cassette in the first cylinder is 12 hours, the dehydration time in the second cylinder is 4 hours, the dehydration time in the third and fourth cylinders is 6 hours, the dehydration time in the fifth to seventh cylinders is 20 minutes, the dehydration time in the eighth and ninth cylinders is 10 minutes, the dehydration time in the tenth cylinder is 1 hour and 30 minutes, the dehydration time in the eleventh and 12 cylinders is 1 hour, and the total dehydration time of the embedding cassette in the tissue dehydrator is 32 hours and 50 minutes.

[0023] Advantages of the invention:

[0024] The method is simple in steps and easy to operate. The intestinal tissue after embedding can maintain morphological integrity during subsequent sectioning, greatly reducing the incidence of separation between the muscular layer and the mucosal layer of the mouse intestine; the connective tissue morphology of the submucosal layer is fully presented, which is convenient for scientific researchers to comprehensively observe the wax block after sectioning, and provides reliable materials for theoretical research on intestinal diseases. Description of the drawings:

[0026] Figure 1 This is an image of the separation of the muscle layer and the mucosal layer under a microscope after the wax block of the intestinal tissue of mice was sliced ​​using the existing technology. A in the figure is the colon image, and B is the rectum image;

[0027] Figure 2 It is a schematic diagram of the structure after completing step B;

[0028] Figure 3 is a schematic diagram of the wax block after it is completely solidified in step D;

[0029] Figure 4 is a schematic diagram after completing step D;

[0030] Figure 5 The following are images of the wax block prepared by the present invention after slicing under a microscope, in which A is a colon image and B is a rectal image;

[0031] Figure 6 The figure is a comparative schematic diagram of the qualified rate after slicing of the wax block prepared by the present invention and the wax block prepared by conventional means. Specific implementation method:

[0033] like Figure 2-6 As shown, a method for preparing a wax block of mouse intestinal tissue for slicing comprises the following steps: Step A is sampling intestinal tissue, which comprises:

[0034] (1) The mice were killed by cervical dislocation and the muscles were cut approximately 2 cm below the sternal angle. The incision was enlarged up and down along the midline to expose the entire intestinal tract.

[0035] (2) Cut the intestine at the pyloric opening of the stomach and the pubic symphysis 1-2 cm above the anus.

[0036] (3) Remove the entire intestinal tract and remove the glands and mesenteric fat attached to the intestinal wall.

[0037] (4) Rinse the intestine with saline and classify it into jejunum, ileum, colon, and rectum. Cut each class into 1 cm long pieces and fix them in formalin for 12-16 hours.

[0038] Step B: Fixation of intestinal tissue. This step includes: (1) taking out the embedding box 1, and pre-laying a first filter paper layer 2 (slightly smaller than the size of the embedding box) on the bottom of the embedding box; and then laying three first non-woven fabric layers 3 of the same size on the filter paper.

[0039] A section of intestinal tissue 4 is taken out from the formalin, placed on the top first non-woven fabric layer 3, and then the intestinal tissue is cut open along the middle using surgical scissors, taking care to avoid damaging the intestinal tissue. The intestinal tissue is transferred to the middle of the non-woven fabric; and the two clamp arms of the forceps are used to press the two ends of the intestinal tissue respectively, so that the intestinal tissue is spread into a rectangular shape and laid flat on the surface of the top first non-woven fabric layer.

[0040] (2) Three layers of second non-woven fabric 5 of the same size are covered on the flattened intestinal tissue 4, and then a second filter paper layer 6 is covered on top of the second non-woven fabric 5. Then, the filter paper is pressed with the index finger instead of tweezers to flatten the intestinal tube, and the tweezers are pulled out of the non-woven fabric to form an intestinal tissue sandwich.

[0041] (3) Use forceps to clamp the prepared intestinal tissue sandwich, and be careful to clamp the intestinal tissue 4 to prevent the intestinal tissue 4 from rebounding into a cylindrical shape. Use a stapler to staple the intestinal tissue sandwich around; the stapler 7 should be located about 8 mm away from the edge of the intestinal tissue 4, because if it is too close to the intestinal tissue 4, the villi of the intestinal tissue 4 will be squeezed, and if it is too far away, the intestinal tissue 4 will rebound into a cylindrical shape.

[0042] The bound intestinal sandwich is placed in an embedding box 1 made of tissue plastic; an anti-pressure layer 8 is covered on top, the anti-pressure layer 8 is made of rubber or PVC, and then the embedding box is covered. The anti-pressure layer 7 is deformed in the direction of the embedding box cover 1a to press the intestinal tissue sandwich below to prevent it from moving in the embedding box 1.

[0043] Since non-woven fabrics are not easy to adhere to intestinal tissues and are water-permeable and breathable, they can protect intestinal tissues during the dehydration process while allowing them to fully contact the dehydration reagents. When only one layer of non-woven fabric is covered, the test found that the intestinal tube cannot be kept flat (the intestinal tube is prone to curling); when two layers are covered, only part of the intestinal tube can be kept flat, and the effect is not stable enough; therefore, when three layers are covered, it is found that almost all of the intestinal tubes can be kept flat.

[0044] (4) Rinse the embedding cassette with running water for 20 min;

[0045] Step C includes placing the embedding cassette into a tissue dehydrator for dehydration, and the entire dehydration process is as follows: the embedding cassette is dehydrated in the first cylinder for 12 hours, and the ethanol concentration in the first cylinder is 50%. The dehydration time in the second cylinder is 4 hours, and the ethanol concentration in the second cylinder is 70%. The dehydration time in the third and fourth cylinders is 6 hours, and the ethanol concentration in the third cylinder is 80%, and the ethanol concentration in the fourth cylinder is 95%. The dehydration time in the fifth to seventh cylinders is 20 minutes, and the ethanol concentration in the fifth to seventh cylinders is 100%.

[0046] The dehydration time in the 8th and 9th cylinders is 10 minutes, and xylene is placed in the 8th and 9th cylinders. The dehydration time in the 10th cylinder is 1 hour and 30 minutes, the dehydration time in the 11th and 12th cylinders is 1 hour, and the slice paraffin is placed in the 10th-12th cylinders. The total dehydration time of the embedding box in the tissue dehydrator is 32 hours and 50 minutes.

[0047] Step D includes injecting a small amount of wax water into the embedding mold 9 so as to cover the bottom surface of the embedding mold 9 with the wax water, then opening the embedding box 1 to take out the dehydrated intestinal tissue sandwich, opening the first and second non-woven fabric layers, using tweezers to take out the intestinal tissue 4, placing the intestinal tissue 4 in the wax water and repeatedly rinsing until obvious bubbles appear on the surface, placing the longitudinal surface of the intestinal tissue 4 in the embedding mold 9 so that it is perpendicular to the bottom surface of the embedding mold 9, moving the embedding mold 9 to a freezing table, and after the wax water at the bottom of the embedding mold 9 solidifies, continuing to add wax water to the embedding mold 9 until the mold is full, and then removing the upper cover of the embedding box 1 and placing the bottom surface of the base on the wax block 10 that is about to solidify. After the wax block is completely solidified, the base of the embedding box 1 is held to take the wax block out of the embedding mold 9, that is, the wax block 10 for slicing is obtained. At this time, the base of the embedding box 1 is located below the wax block 10 and can play the role of a support. Under the microscope, the intestinal tissue after sectioning is shown to be tightly fitted between the muscular layer Z and the mucosal layer X, with no gap Y formed between them.

[0048] like Figure 6 As shown, in order to prove the reliability of the preparation method of the mouse intestinal tissue wax block for slicing provided by this patent, we conducted a comparative experiment of homogeneous specimens. Group A adopted the conventional dehydration embedding method, and Group B adopted the preparation method provided by this application. After slicing (5 μm) and H&E staining, they were uniformly photographed under a microscope (× 200 times), the non-separation length of the muscle layer was measured, and the percentage of the non-separation length to the total length of the intestinal tube was calculated. The non-separation length ratio of the muscle layer ≥80% was identified as a qualified slice.

[0049] pass Figure 6 It can be seen that the method for preparing a mouse intestinal tissue wax block for slicing provided in the present application is a significant improvement over conventional methods.

Claims

1. A method for preparing a wax block of mouse intestinal tissue for slicing, comprising the following steps: Step A: sampling of intestinal tissue, characterized by: step B: fixation of the intestinal tissue, step C: dehydration of the intestinal tissue, and step D: embedding of the intestinal tissue; Step B includes: (1) Take out the embedding box, and pre-lay a first filter paper layer on the bottom of the embedding box; lay at least two first non-woven fabric layers of the same size on the first filter paper layer, cut the intestinal tract in the middle, and transfer the intestinal tissue to the middle position of the non-woven fabric; flatten the intestinal tissue, spread it into a rectangle, and fix it; (2) Cover the flattened intestinal tissue with at least two layers of the second non-woven fabric layer of the same size, and then cover the second filter paper layer on the second non-woven fabric layer, and then press the filter paper with the index finger to flatten the intestinal tissue, thereby forming an intestinal tissue sandwich; (3) Stapling the intestinal interlayer around; placing the stapled intestinal interlayer into a tissue embedding box; covering the intestinal interlayer with a pressure-resistant layer made of rubber or PVC, and then covering the embedding box. The pressure-resistant layer is deformed in the squeezing direction of the embedding box cover to press the intestinal tissue interlayer below to prevent it from moving in the embedding box; (4) Rinse the embedding cassette with running water for 17-25 minutes; Step C includes placing the embedding cassette into a tissue dehydrator for dehydration; Step D includes injecting wax water into the embedding mold, then opening the embedding box to take out the dehydrated intestinal tissue layer, opening the non-woven fabric to take out the intestinal tissue, placing the intestinal tissue in the wax water and repeatedly rinsing until bubbles are obvious on the surface, placing the longitudinal surface of the intestinal tissue on the embedding mold so that it is perpendicular to the bottom surface of the embedding mold, moving the mold to a freezing table, and when the wax water at the bottom of the mold solidifies, continuing to add wax water to the mold until the mold is full, and then placing the embedding box base upside down on the wax block that is about to solidify, and after the wax block is completely solidified, holding the embedding box base to take the wax block out of the embedding mold to obtain a wax block for slicing.

2. The method for preparing a wax block of mouse intestinal tissue for slicing according to claim 1, characterized in that: In step B, the first filter paper layer is covered with three layers of non-woven fabrics, and the intestinal tissue is covered with three layers of non-woven fabrics.

3. The method for preparing a wax block of mouse intestinal tissue for slicing according to claim 1, characterized in that: In step B, a stapler is used to staple the intestinal tissue layer in the thickness direction, and the stapler position needs to be 6-10 mm away from the edge of the tissue.

4. The method for preparing a wax block of mouse intestinal tissue for slicing according to claim 1, characterized in that: In the cylinder group of the tissue dehydrator in step C, ethanol is placed in cylinders 1 to 7, xylene is placed in cylinders 8 and 9, and slice paraffin is placed in cylinders 10 to 12.

5. The method for preparing a wax block of mouse intestinal tissue for slicing according to claim 4, characterized in that: The ethanol concentration in the first cylinder is 50%, the ethanol concentration in the second cylinder is 70%, the ethanol concentration in the third cylinder is 80%, the ethanol concentration in the fourth cylinder is 95%, and the ethanol concentrations in the fifth to seventh cylinders are 100%.

6. A method for preparing a wax block of mouse intestinal tissue for slicing according to claim 5 or 1, characterized in that: The dehydration time of the embedding cassette in the first cylinder is 12 hours, the dehydration time in the second cylinder is 4 hours, the dehydration time in the third and fourth cylinders is 6 hours, the dehydration time in the fifth to seventh cylinders is 20 minutes, the dehydration time in the eighth and ninth cylinders is 10 minutes, the dehydration time in the tenth cylinder is 1 hour and 30 minutes, the dehydration time in the eleventh and 12 cylinders is 1 hour, and the total dehydration time of the embedding cassette in the tissue dehydrator is 32 hours and 50 minutes.

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