CN112067382A - Preparation method of small intestine slices - Google Patents
Preparation method of small intestine slices Download PDFInfo
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- CN112067382A CN112067382A CN202010788392.8A CN202010788392A CN112067382A CN 112067382 A CN112067382 A CN 112067382A CN 202010788392 A CN202010788392 A CN 202010788392A CN 112067382 A CN112067382 A CN 112067382A
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- small intestine
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- cylinder
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- 210000000813 small intestine Anatomy 0.000 title claims abstract description 86
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 230000000968 intestinal effect Effects 0.000 claims abstract description 21
- 210000000713 mesentery Anatomy 0.000 claims abstract description 9
- 210000000936 intestine Anatomy 0.000 claims abstract description 7
- 238000002791 soaking Methods 0.000 claims abstract description 6
- 238000005406 washing Methods 0.000 claims abstract description 6
- 210000004204 blood vessel Anatomy 0.000 claims abstract description 4
- 238000010186 staining Methods 0.000 claims abstract description 4
- 238000004804 winding Methods 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims abstract description 3
- 238000007493 shaping process Methods 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 8
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 229920001223 polyethylene glycol Polymers 0.000 claims description 5
- -1 ethanol-polyethylene Chemical group 0.000 claims description 4
- 239000000834 fixative Substances 0.000 claims 2
- 230000000052 comparative effect Effects 0.000 description 5
- 239000011521 glass Substances 0.000 description 4
- 210000004877 mucosa Anatomy 0.000 description 3
- 210000004400 mucous membrane Anatomy 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000000635 electron micrograph Methods 0.000 description 2
- 210000003736 gastrointestinal content Anatomy 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NLMDJJTUQPXZFG-UHFFFAOYSA-N 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane Chemical compound C1COCCOCCNCCOCCOCCN1 NLMDJJTUQPXZFG-UHFFFAOYSA-N 0.000 description 1
- 239000011547 Bouin solution Substances 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
Abstract
The invention provides a preparation method of small intestine slices, which comprises the following specific steps: (1) carrying out blunt separation on mesentery of the small intestine, removing blood vessels and residual fat masses, and then taking out the content of the small intestine; (2) after the content of the small intestine is taken out, the intestinal wall is cut along the longitudinal direction of the mesentery, so that the intestinal wall is turned outwards, and the mucous layer of the intestine is fully exposed; (3) fixing the cut small intestine on a plate, then immersing the small intestine in the Taiwanese liquid, and placing the small intestine in a thermostat to wash the intestinal cavity; (4) taking out the washed small intestine from the Taiwanese liquid, and putting the small intestine into a fixing liquid for soaking and fixing; (5) taking the cylinder as an axis, winding the outer wall of the soaked and fixed small intestine inwards and the inner wall outwards on the cylinder for shaping; (6) and taking the shaped small intestine down from the cylinder, and then sequentially washing, dehydrating, embedding, slicing along the radial direction and staining. The small intestine slice prepared by the preparation method has a complete structure, the structure of the small intestine slice is not easy to damage, and the preparation method is time-saving and labor-saving.
Description
Technical Field
The invention belongs to the field of experimental materials, and particularly relates to a preparation method of a small intestine slice.
Background
The small intestine is the main part of digestion and absorption, the cavity surface is an annular fold, the mucous membrane surface has a plurality of fine intestinal villi, and pathological sections are often needed to be carried out on the small intestine in the pathological histological observation so as to observe the influence of diseases or medicaments on the structure of the small intestine, the length of the intestinal villi, the height of crypt and the like; when the traditional method is used for manufacturing a small intestine tissue teaching section, the tissue is easy to crack, the cells shrink, the section is uneven in thickness, and the staining is too deep or too shallow, so that the chromatin structure is not clearly observed under a microscope.
Disclosure of Invention
The present invention is directed to a method for preparing a small intestine slice, which solves one or more of the problems of the prior art, and provides at least one of the advantages of the present invention.
In order to realize the purpose, the technical scheme is as follows:
a preparation method of small intestine slices comprises the following specific steps:
(1) carrying out blunt separation on mesentery of the small intestine, removing blood vessels and residual fat masses, and then taking out the content of the small intestine;
(2) after the content of the small intestine is taken out, the intestinal wall is cut along the longitudinal direction of the mesentery, so that the intestinal wall is turned outwards, and the mucous layer of the intestine is fully exposed;
(3) fixing the cut small intestine on a plate, then immersing the small intestine in the Taiwanese liquid, and placing the small intestine in a thermostat to wash the intestinal cavity;
(4) taking out the washed small intestine from the Taiwanese liquid, and putting the small intestine into a fixing liquid for soaking and fixing;
(5) taking the cylinder as an axis, winding the outer wall of the soaked and fixed small intestine inwards and the inner wall outwards on the cylinder for shaping;
(6) and taking the shaped small intestine down from the cylinder, and then sequentially washing, dehydrating, embedding, slicing along the radial direction and staining.
The addition amount of the typhoid liquid in the step (3) is 450-600 mL.
The temperature of the constant temperature box in the step (3) is 37-39 ℃.
The time for flushing in the step (3) is 25-35 min.
The stationary liquid is 95% ethanol-polyethylene glycol solution.
The addition amount of the fixing liquid is 220-350 mL.
The soaking time is 35-52 h.
The invention has the beneficial effects that: the small intestine section is radially sliced after being fixed by taking a cylinder as an axis, so that a longitudinal section of the whole intestine section can be made, and the specific structure can be observed conveniently.
The fixing solution added in the preparation method is a 95% ethanol-polyethylene glycol solution, wherein ethanol and polyethylene glycol are alcohols and are volatile, but the fixing solution is easier to obtain compared with formaldehyde, paraformaldehyde and bouin's solution contained in a common fixing solution, is nontoxic to human bodies, does not stimulate respiratory tracts and mucous membranes, has better fixation effect on cell nuclei and clear structure, and can also protect cells from artificial artifacts after air drying.
Drawings
FIG. 1 is an electron micrograph of a section of a small intestine prepared in a comparative example.
FIG. 2 is an electron micrograph of a section of a small intestine prepared in example 1.
Detailed Description
The following steps are only used for illustrating the technical scheme of the invention and are not limited; although the present invention has been described in detail with reference to the foregoing steps, it will be understood by those of ordinary skill in the art that: the technical solutions recorded in the foregoing steps may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the present invention in its various steps.
Example 1
A preparation method of small intestine slices comprises the following specific steps:
(1) taking out small intestine, separating mesentery, and removing blood vessel and residual fat block with surgical scissors;
(2) some intestinal contents exist in the small intestine, and need to be taken out, otherwise the slicing quality is influenced, the intestinal wall is longitudinally cut along the mesentery end, so that the intestinal wall is outwards turned out, and the mucosa layer of the intestine is fully exposed;
(3) fixing the four corners of the cut small intestine on a hardboard by using a pin, immersing the small intestine in 500mL of Taiwan liquid, placing the small intestine in a thermostat at 38 ℃ for 30 minutes, and continuously sucking the Taiwan liquid by using a 1000-volume micro liquid transfer gun to flush the intestinal cavity;
(4) after no intestinal content is observed, taking out the intestines from the table liquid, and soaking in 300mL of 95% ethanol-polyethylene glycol solution for 48 hours;
(5) the outer wall of the small intestine after being soaked and fixed is inward, the inner wall is outward wound on a wooden cylinder with the length of 5 cm and the width of 0.4 cm, and the small intestine is shaped into an intestinal roll shape;
(6) taking down the shaped small intestine from the cylinder, dividing the small intestine into two sections from the middle by taking care of the mucosa layer which does not damage the intestine, and putting the sections into an embedding frame for washing, dehydrating, embedding, slicing along the radial direction and dyeing in sequence.
Comparative example
A preparation method of small intestine slices comprises the following specific steps:
(1) placing the small intestine in the bottom or cover of a culture dish, placing some normal saline, taking a 10 ml syringe (provided with a coarse needle with a flat head), sucking normal saline (room temperature), and washing the intestinal lumen;
(2) placing the washed small intestine on a rectangular wood board (with the length of about 80 cm, the width and the thickness of the small intestine are not limited) with a layer of filter paper (crude, pre-soaked by water) laid on the surface, cutting the small intestine longitudinally along the mesentery attachment line, and spreading the intestinal wall on the filter paper by using a self-made L-shaped thin glass rod, wherein the mucous membrane surface is naturally upward;
(3) taking a 5 ml syringe, sleeving a thin needle with a polished tip, filling Boitun's stationary liquid in the syringe, spraying the stationary liquid on a mucosal surface, and standing in the air for 10-20 minutes;
(4) after Boitun's stationary liquid is sprayed and the small intestine is stood, the small intestine is coiled on a serial number of fixing plates made of organic glass with the mucosa facing outwards, then the fixing plates are placed in a rectangular glass jar with a fixing frame and containing Bouin's stationary liquid for full fixation, and the standing is carried out for 24 hours;
(5) washing with running water for 24h after standing, taking down the small intestine, winding the whole small intestine on a glass rod gradually from one end to shape, taking down the shaped small intestine, dehydrating, embedding, transversely slicing, and dyeing.
The small intestine slices prepared in example 1 and comparative example are respectively placed under an electron microscope for observation, and the results are shown in fig. 1 and fig. 2, fig. 1 is an electron microscope image of the small intestine slice prepared in the comparative example, the intestinal villi can be easily damaged by the preparation method of the small intestine slice described in the comparative example can be seen from fig. 1-a, fig. 1-B can be seen that the intestinal villi can be shed and the small intestine slice is broken by the preparation method of the small intestine slice described in example 1, fig. 2 is an electron microscope image of the small intestine slice prepared in the preparation method of the small intestine slice described in example 1, wherein fig. 2-a is a small intestine slice image under a 100-fold microscope, it can be seen that the intestinal fixed layer and crypt of the small intestine slice prepared by the preparation method of the small intestine slice described in the invention are complete, fig. 2-B is a small intestine slice image under a 400-fold microscope, it can be seen that the small intestine fixed layer and crypt of the small intestine slice prepared by the, this facilitates subsequent measurements.
Therefore, the Taiwanese liquid used in the preparation method can maintain the nutrition and the shape of intestinal villi, is beneficial to discharging contents, and ensures that the prepared small intestine slice is clear and free of impurities.
Claims (7)
1. The preparation method of the small intestine slice is characterized by comprising the following specific steps:
(1) carrying out blunt separation on mesentery of the small intestine, removing blood vessels and residual fat masses, and then taking out the content of the small intestine;
(2) after the content of the small intestine is taken out, the intestinal wall is cut along the longitudinal direction of the mesentery, so that the intestinal wall is turned outwards, and the mucous layer of the intestine is fully exposed;
(3) fixing the cut small intestine on a plate, then immersing the small intestine in the Taiwanese liquid, and placing the small intestine in a thermostat to wash the intestinal cavity;
(4) taking out the washed small intestine from the Taiwanese liquid, and putting the small intestine into a fixing liquid for soaking and fixing;
(5) taking the cylinder as an axis, winding the outer wall of the soaked and fixed small intestine inwards and the inner wall outwards on the cylinder for shaping;
(6) and taking the shaped small intestine down from the cylinder, and then sequentially washing, dehydrating, embedding, slicing along the radial direction and staining.
2. The method for preparing a small intestine section as defined in claim 1, wherein the amount of the Taiwanese liquid added in step (3) is 450-600 mL.
3. The method for preparing a small intestine slice according to claim 1, wherein the temperature of the incubator in the step (3) is 37 to 39 ℃.
4. The method for preparing a small intestine slice according to claim 1, wherein the time for rinsing in step (3) is 25-35 min.
5. The method of preparing a small intestine slice according to claim 1, wherein the fixative is a 95% ethanol-polyethylene glycol solution.
6. The method for preparing a small intestine section as defined in claim 1, wherein the amount of the fixative is 220-350 mL.
7. The method for preparing a small intestine slice according to claim 1, wherein the soaking is carried out for a fixed period of time of 35-52 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010788392.8A CN112067382A (en) | 2020-08-07 | 2020-08-07 | Preparation method of small intestine slices |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010788392.8A CN112067382A (en) | 2020-08-07 | 2020-08-07 | Preparation method of small intestine slices |
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| CN112067382A true CN112067382A (en) | 2020-12-11 |
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| CN202010788392.8A Pending CN112067382A (en) | 2020-08-07 | 2020-08-07 | Preparation method of small intestine slices |
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-
2020
- 2020-08-07 CN CN202010788392.8A patent/CN112067382A/en active Pending
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