A mouse intestinal pathological section embedding box and a method for making pathological sections

Abstract

A mouse intestinal pathological section embedding box is provided with an anti-collision block for fixing a Swiss roll, two non-woven fabric sheets, and a punching assembly for punching the Swiss roll. When in operation, the Swiss roll is fixed by the anti-collision block and the two non-woven fabric sheets, so that gaps are not easily generated in the formed wax block, and the subsequent sectioning and staining are more favorable, and the intestinal canal is not damaged. The method for making pathological sections comprises the following steps: separating the matched tool box from the embedding box body, punching the anti-collision block with a suitable punch, cleaning and rolling the mouse intestinal canal fixed by formalin into a Swiss roll; inserting the Swiss roll into the hole by using external force and flattening the protruding part of the intestinal canal tissue with a blade, placing the two layers of non-woven fabric to clamp the anti-collision block into the embedding box for tissue dehydration, peeling off the Swiss roll from the anti-collision block after dehydration and making a wax block; freezing and re-solidifying after heating and melting the paraffin, and then sectioning. The method can improve the overall morphological quality of pathological sections, and there is no phenomenon of structural loss.

Description

TECHNICAL FIELD

[0001] The present application relates to a mouse intestinal pathological section embedding box and a method for making pathological sections. BACKGROUND

[0002] The mouse intestinal "Swiss roll" pathological section is an important means for studying intestinal diseases. The specific process is described in the reference LeNaour J, Montegut L, Joseph A, Garbin K, Vacchelli E, Kroemer G, Pol JG, Maiuri MC. Improved Swiss-rolling method for histological analyses of colon tissue. Methods X. 2022 Feb 9; 9: 101630. doi: 10.1016 / j.mex.2022.101630. PMID: 35242614; PMCID: PMC8861817. However, due to various constraints, the current technology still has the following problems: 1) The mouse intestinal "Swiss roll" pathological section cannot fully show the shape and appearance of the whole intestinal canal; 2) When rolled to the end, a thin needle is needed for fixation, which can easily cause mechanical damage to the intestinal canal tissue; 3) After dehydration, the intestinal canal is easy to spread open, and the overall morphology is difficult to maintain, which is not conducive to subsequent histological analysis; 4) The submucosal layer and muscle layer of the intestinal wall are easy to separate. SUMMARY

[0003] The first technical problem to be solved by the present application is to provide a mouse intestinal pathological section embedding box, which is provided with an anti-collision block for fixing the Swiss roll, two non-woven fabric sheets, and a punching assembly for punching the Swiss roll. When operating, the Swiss roll can be fixed by the anti-collision block and the two non-woven fabric sheets, so that the gap in the formed wax block is not easy to produce, which is more conducive to subsequent sectioning and staining, and will not cause damage to the intestinal canal.

[0004] Another technical problem to be solved by the present application is to provide a method for making pathological sections using the above-mentioned mouse intestinal pathological section embedding box. The method has simple steps and high experimental success rate. The method can significantly improve the overall morphology quality of the mouse intestinal pathological section, improve the separation of the submucosal layer and muscle layer, and protect the structural integrity of the whole intestinal canal and the whole intestinal canal.

[0005] To solve the above-mentioned first technical problem, the present application provides a mouse intestinal pathological section embedding box, which comprises an embedding box body and a matched tool box.

[0006] The embedding box body cavity is provided with a first non-woven fabric sheet, an NBR material anti-collision block and a second non-woven fabric sheet which are stacked from bottom to top, the NBR material anti-collision block has a size of 25mm in length, 35mm in width and 3-4mm in thickness, the first non-woven fabric sheet and the second non-woven fabric sheet have the same size of 25mm in length, 35mm in width and 0.1-0.2mm in thickness, and a magnetic steel is pre-embedded on the left side or the right side of the side edge of the embedding box body;

[0007] The matched tool box is a long strip box body which is horizontally arranged and has a front-to-back length direction, a flip cover is hingedly connected to the upper side of the long strip box body, the matched tool box is attached to the side edge of the embedding box body which is provided with the magnetic steel, a clapper which is matched with the magnetic steel is pre-embedded at the position of the magnetic steel of the embedding box body, and the matched tool box is adsorbed to the side edge of the embedding box body through the cooperation of the clapper and the magnetic steel.

[0008] The matched tool box is provided with a punching assembly, the punching assembly comprises a positioning rod, a plurality of opening devices with different diameters and a shifting rod, the positioning rod is a round rod, an outer thread is arranged on the circumferential outer wall of the length direction side of the positioning rod, the opening device is a cylindrical body, the cylindrical body is open at one axial end and is sealed at the other end, a sawtooth with an open blade is arranged on the circumferential side of the open end of the cylindrical body, a threaded hole which is matched with the outer thread of the positioning rod is arranged at the center of the sealed end of the cylindrical body, the axial length of each opening device is smaller than the circumferential length of the outer thread, a radial through hole which penetrates the circumferential side wall of the opening device in the radial direction is arranged on the circumferential side of each opening device, and the shifting rod is a long strip rod body, the diameter of the shifting rod is matched with the radial through hole.

[0009] For the sake of simplicity, the mouse intestinal pathological section embedding box described in the present application is simply referred to as the embedding box.

[0010] The embedding box has the advantages that the anti-collision block, the two non-woven fabric sheets and the punching assembly for punching the Swiss roll are matched in the embedding box, the Swiss roll is fixed through the anti-collision block and the two non-woven fabric sheets, so that the gap in the formed wax block is not easy to be generated, which is more conducive to subsequent slicing and dyeing, the anti-collision block made of NBR material is a material with good flexibility, so that the intestinal tube is uniformly stressed and is not damaged, and the anti-collision block can keep a more stable shape during dehydration. In addition, the existing method is easy to cause the separation of the submucosa and the muscle layer, and the anti-collision block in the embedding box can reduce the probability of separation. The embedding box is provided with a plurality of opening devices with different diameters, so that the opening device with a suitable diameter can be selected to punch the anti-collision block according to the needs, a large number of experiments show that the ileum generally selects a hole diameter of 3.0-3.5mm, and the colon and rectum generally selects a hole diameter of 2.5-3.0mm.

[0011] To solve the above-mentioned second technical problem, the application provides a pathological section embedding box for pathological section of mouse intestinal tract, and the method comprises the following steps:

[0012] (1) Separate the matched tool box from the embedding box body, open the embedding box body, take out the first non-woven fabric sheet, the anti-collision block and the second non-woven fabric sheet, open the matched tool box to take out the positioning rod, the hole opener and the push rod, select the hole opener with a proper diameter according to the requirement, vertically arrange the threaded end of the positioning rod downward, and rotate the opening end of the hole opener with the positioning rod downward, so that the lower end of the positioning rod exceeds the lower end of the hole opener, then place the anti-collision block horizontally on the table top, and press the center of the anti-collision block with the lower end of the positioning rod, at this time, insert one end of the push rod into the radial through hole of the hole opener, push the hole opener to rotate and move downward around the positioning rod, and a circular hole is cut in the anti-collision block by the blade saw teeth of the hole opener during the rotating and moving downward of the hole opener;

[0013] (2) Take the mouse intestine fixed by formalin, and remove the mesenteric fat and vascular tissue attached to the surface of the intestine;

[0014] (3) Open the whole intestine along the length direction of the wall of the intestine, and clean the feces in the intestine by using PBS;

[0015] (4) After the cleaning of the intestine, hold the proximal end of the intestine with a fine forceps, and slowly wind the remaining intestine around the proximal end of the intestine as the center axis to form a cylindrical Swiss roll;

[0016] (5) Stretch and expand the circular hole by applying external force to the anti-collision block horizontally and outward, then insert the cylindrical Swiss roll into the circular hole with the end surface downward, so that the axial both ends of the Swiss roll exceed the thickness direction of both sides of the anti-collision block, then release the anti-collision block to restore the original state, then take out the fine forceps, and at this time, the Swiss roll is tightly matched with the circular hole;

[0017] (6) Cut the part of the intestine tissue exceeding the anti-collision block with a blade, and cut the axial both ends of the Swiss roll to be flush with the thickness direction of both sides of the anti-collision block;

[0018] (7) Place the first non-woven fabric sheet in the embedding box to lay on the bottom of the box, then place the anti-collision block with the Swiss roll on the first non-woven fabric sheet, then cover the second non-woven fabric sheet on the upper side of the anti-collision block, and finally cover the embedding box body upper cover to dehydrate the tissue;

[0019] (8) After dehydration, open the embedding box and take out the anti-collision block, then separate the Swiss roll from the anti-collision block, then repeatedly rinse the surface with wax water, and place the Swiss roll on the bottom of the embedding mold to pour the wax water to form a wax block; place the solidified wax block in a 70℃ vacuum drying box for 3-4h, take out the wax block after the paraffin is completely melted, place the wax block on the freezing table again to make it solidify again, and then the sectioning can be performed.

[0020] For the sake of simplicity, the following method for making pathological sections of a mouse intestinal pathological section embedding box described in the present application is referred to as the present method.

[0021] The advantages of the present method are: H&E staining shows that, compared with the conventional method group, the overall shape of the pathological sections made by the present method is regular, mostly circular, the intestinal wall structure does not show damage caused by instruments, and the submucosal layer and muscle layer are rarely separated, which is conducive to the observation of the integrity of the intestine; immunohistochemical analysis shows that the blood vessels and lymphatic vessels in the submucosal layer of the mouse intestine made by the present method are more complete, which provides conditions for exploring the pathological changes and influencing factors of the submucosal layer of the intestinal disease; immunofluorescence staining shows that the pathological sections made by the present method can observe the distribution of Paneth cells and goblet cells in the entire intestinal canal in the mouse ileum and colorectum, and there is no phenomenon of structural loss; the present method has the advantages of strong operability, strong repeatability, stable effect, and the like, and the materials involved are cheap and easy to obtain. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a top view of the embedding box.

[0023] Figure 2 is a front view of the embedding box.

[0024] Figure 3 is Figure 2 a partial enlarged view of part A.

[0025] Figure 4 is Figure 2 a B-B sectional view of

[0026] Figure 5 is a structural schematic view of the positioning rod in the matching tool box.

[0027] Figure 6 is a structural schematic view of the hole opener in the matching tool box (all hole openers have the same structure except for the hole diameter).

[0028] Figure 7 is a schematic view of the punching assembly in use.

[0029] Figure 8 is another schematic view of the punching assembly in use.

[0030] Figure 9 is a practical operation photo of step (2) in the present embodiment.

[0031] Figure 10 is a practical operation photo of step (3) in the present embodiment.

[0032] Figure 11is the actual operation photo of step (4) in this embodiment.

[0033] Figure 12 is the actual operation photo of step (5) in this embodiment.

[0034] Figure 13 is the actual operation photo of step (6) in this embodiment.

[0035] Figure 14 is the actual operation photo of step (7) in this embodiment.

[0036] Figure 15 is the actual operation photo of step (8) in this embodiment. DETAILED DESCRIPTION

[0037] Referring to Figures 1-8 A mouse intestinal pathological section embedding box comprises an embedding box body 1 and a matched tool box 2.

[0038] The inner cavity of the embedding box body 1 is provided with a first non-woven fabric sheet 3, an NBR material anti-collision block 31 and a second non-woven fabric sheet 32 which are stacked in sequence from bottom to top. The NBR material anti-collision block 31 has a size of 25 mm in length, 35 mm in width and 3-4 mm in thickness. The first non-woven fabric sheet 3 and the second non-woven fabric sheet 32 have the same size, i.e. 25 mm in length, 35 mm in width and 0.1-0.2 mm in thickness. A magnetic steel 4 is pre-embedded on the left or right side of the embedding box body 1.

[0039] The matched tool box 2 is a long strip-shaped box body which is horizontally arranged and has a front-to-back length direction. A flip cover is hingedly connected to the upper side of the long strip-shaped box body. The matched tool box 2 is attached to the side of the embedding box body on which the magnetic steel 4 is arranged. A clapper 41 matched with the magnetic steel 4 is pre-embedded on the side of the matched tool box 2 corresponding to the magnetic steel 4 of the embedding box body 1. The matched tool box 2 is adsorbed to the side of the embedding box body 1 through the cooperation of the clapper 41 and the magnetic steel 4.

[0040] The matched tool box 2 is provided with a punching assembly. The punching assembly comprises a positioning rod 5, a plurality of hole openers 6 with different diameters and a shifting rod 7. The positioning rod 5 is a circular rod. An outer thread is arranged on the circumferential outer wall of one side of the positioning rod 5 in the length direction. The hole opener 6 is a cylindrical body. The cylindrical body is open at one axial end and sealed at the other end. A sawtooth 63 with an open blade is arranged on the circumferential side of the open end of the cylindrical body. A threaded hole 61 matched with the outer thread of the positioning rod 5 is arranged at the center of the sealed end of the cylindrical body. The axial length of each hole opener 6 is smaller than the circumferential length of the outer thread. A radial through hole 62 penetrating the circumferential side wall of the hole opener 6 in the radial direction is arranged on the circumferential side of each hole opener 6. The shifting rod 7 is a long strip-shaped rod body. The diameter of the shifting rod 7 is matched with the radial through hole 62.

[0041] The embedding box body part in the embodiment has no structural difference from the existing commercially available embedding box (only a magnetic steel is embedded at the side edge of one side), so it is not described in detail in the application.

[0042] A method for making pathological sections of mouse intestinal pathological sections, comprising the following steps:

[0043] (1) Separate the matched tool box from the embedding box body, open the embedding box body, take out the first non-woven fabric sheet, the anti-collision block and the second non-woven fabric sheet, open the matched tool box to take out the positioning rod, the hole opener and the push rod, select the hole opener with the appropriate caliber according to the needs, vertically arrange the threaded end of the positioning rod downward, and rotate the opening end of the hole opener with the positioning rod downward, so that the lower end of the positioning rod exceeds the lower end of the hole opener, then place the anti-collision block flat on the table top, and use the lower end of the positioning rod to abut against the center of the anti-collision block. At this time, one end of the push rod is inserted into the radial through hole of the hole opener, and the hole opener is pushed by the push rod to rotate downward around the positioning rod. During the rotation and downward movement of the hole opener, the anti-collision block is cut by the blade saw teeth of the hole opener to form a circular hole;

[0044] (2) Referring to Figure 9 , take the mouse intestine fixed by formalin, and remove the mesenteric fat and vascular tissue attached to the surface of the intestine;

[0045] (3) Referring to Figure 10 , cut the entire intestine along the length direction of the intestinal wall, and clean the feces in the intestine using PBS;

[0046] (4) Referring to Figure 11 , after the cleaning of the intestine is completed, hold the proximal end of the intestine with a fine forceps in one hand, and slowly wrap the remaining intestine around the proximal end of the intestine as the center axis to form a cylindrical Swiss roll with the other hand;

[0047] (5) Referring to Figure 12 , expand the hole diameter of the circular hole by applying an external force to stretch the anti-collision block horizontally and outward, then insert the cylindrical Swiss roll end face downward into the circular hole, so that the axial both ends of the Swiss roll exceed the thickness direction of both sides of the anti-collision block, then release the anti-collision block to restore its original state, then pull out the fine forceps, and at this time the Swiss roll is tightly matched with the circular hole;

[0048] (6) Referring to Figure 13 , cut the part of the intestine tissue exceeding the anti-collision block with a blade, and cut the axial both ends of the Swiss roll to be flush with the two sides of the thickness direction of the anti-collision block respectively;

[0049] (7) Referring to Figure 14Put the first non-woven fabric sheet into the embedding box and lay it flat on the bottom of the box, then lay the anti-collision block with the Swiss roll on the first non-woven fabric sheet, then cover the second non-woven fabric sheet on the upper side of the anti-collision block, and finally cover the upper cover of the embedding box body to dehydrate the tissue;

[0050] (8) After dehydration, open the embedding box and take out the anti-collision block, then peel off the Swiss roll from the anti-collision block, then repeatedly rinse the surface with wax water, and place it at the bottom of the embedding mold, pour wax water to make a wax block; after the wax block solidifies, it is placed in a 70℃ vacuum drying box for 3-4h, then taken out after the paraffin is completely melted, placed on the freezing table again to solidify, and then slicing can be performed.

Claims

1. A method for preparing pathological sections using a mouse intestinal pathological section embedding cassette, characterized in that: The embedding box includes an embedding box body and a matching tool box; The embedding cassette body has a first non-woven fabric sheet, an NBR anti-collision block, and a second non-woven fabric sheet stacked from bottom to top in its internal cavity. The NBR anti-collision block is 25mm long, 35mm wide, and 3-4mm thick. The first and second non-woven fabric sheets are the same size, both 25mm long, 35mm wide, and 0.1-0.2mm thick. A magnet is pre-embedded on the left or right side of the embedding cassette body. The toolbox is a long, rectangular box arranged horizontally and running back and forth. A hinged flap is attached to the top of the box. The toolbox is attached to the side of the embedding box where the magnet is located. An armature matching the magnet is pre-embedded on the side of the toolbox, corresponding to the magnet on the embedding box. The toolbox is attracted to the side of the embedding box by the cooperation of the armature and the magnet. The toolbox contains a drilling assembly, which includes a positioning rod, several hole openers of different diameters, and a lever. The positioning rod is a round rod with external threads on its circumferential outer wall along its length. The hole opener is a cylindrical body with one open end and the other closed. The open end of the cylindrical body has serrated edges along its circumference, and the center of the closed end of the cylindrical body has a threaded through hole that matches the external threads on the positioning rod. The axial length of each hole opener is less than the circumferential length of the external threads. Each hole opener has a radial through hole on its circumferential side that penetrates the circumferential side wall of the hole opener. The lever is a long strip rod with a diameter that matches the radial through hole. The steps for preparing pathological sections are as follows: (1) Separate the tool box from the embedding box body, open the embedding box body, take out the first non-woven fabric piece, the anti-collision block, and the second non-woven fabric piece, open the tool box and take out the positioning rod, the hole opener and the lever. Select the appropriate diameter hole opener as needed, arrange the threaded end of the positioning rod vertically downward, and screw the opening end of the hole opener downward to the positioning rod so that the lower end of the positioning rod extends beyond the lower end of the hole opener. Then place the anti-collision block flat on the table and use the lower end of the positioning rod to hold the center of the anti-collision block. At this time, insert one end of the lever into the radial through hole of the hole opener and push the hole opener to rotate and move downward around the positioning rod. During the rotation and downward movement of the hole opener, the anti-collision block is cut into a circular hole by the saw teeth of the hole opener. (2) Take the mouse intestine after it has been fixed in formalin and remove the mesenteric fat and blood vessel tissue attached to the surface of the intestine; (3) The entire intestinal segment was cut open along the length of the intestinal wall and the feces inside the intestinal segment were washed with PBS; (4) After the intestine is cleaned, hold the proximal end of the intestine with fine tweezers in one hand, and slowly wrap the remaining intestine with the proximal end of the intestine held in the other hand to form a cylindrical Swiss roll. (5) By applying external force, the anti-collision block is stretched horizontally and outward to enlarge the diameter of the circular hole. Then, the cylindrical Swiss roll is inserted into the circular hole with the end facing down, so that both ends of the Swiss roll extend beyond the thickness direction of the anti-collision block. Then, the anti-collision block is released to restore its original shape. Then, the thin tweezers are pulled out. At this time, the Swiss roll and the circular hole fit tightly together. (6) Use a blade to remove the portion of intestinal tissue that extends beyond the anti-collision block. The cut Swiss roll is aligned with the two sides of the anti-collision block in the thickness direction at both ends. (7) Place the first nonwoven fabric sheet into the embedding box and lay it flat on the bottom of the box. Then place the anti-collision block with Swiss roll flat on the first nonwoven fabric sheet. Then cover the second nonwoven fabric sheet on the upper side of the anti-collision block. Finally, cover the upper cover of the embedding box body to dehydrate the tissue. (8) After dehydration, open the embedding box and take out the anti-collision block. Then, separate the Swiss roll from the anti-collision block, rinse the surface repeatedly with wax water, place it at the bottom of the embedding mold, pour wax water to make a wax block; put the solidified wax block into a 70℃ vacuum drying oven for 3-4 hours. After the paraffin wax has completely melted, take it out and put it back on the freezing table to solidify it again, and then it can be sectioned.

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