CN112393962A - Three-dimensional reconstruction method of forest musk gland tissue - Google Patents
Three-dimensional reconstruction method of forest musk gland tissue Download PDFInfo
- Publication number
- CN112393962A CN112393962A CN202011371039.6A CN202011371039A CN112393962A CN 112393962 A CN112393962 A CN 112393962A CN 202011371039 A CN202011371039 A CN 202011371039A CN 112393962 A CN112393962 A CN 112393962A
- Authority
- CN
- China
- Prior art keywords
- slices
- musk
- forest
- 5min
- alcohol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000402754 Erythranthe moschata Species 0.000 title claims abstract description 62
- 210000004907 gland Anatomy 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 19
- 238000010186 staining Methods 0.000 claims abstract description 17
- 238000000338 in vitro Methods 0.000 claims abstract description 12
- 238000005070 sampling Methods 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 86
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 53
- 235000019441 ethanol Nutrition 0.000 claims description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 239000008096 xylene Substances 0.000 claims description 29
- 239000000463 material Substances 0.000 claims description 26
- 239000001993 wax Substances 0.000 claims description 25
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 20
- 239000012188 paraffin wax Substances 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 20
- 241001416180 Moschidae Species 0.000 claims description 18
- 238000007598 dipping method Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 230000004069 differentiation Effects 0.000 claims description 11
- 230000007480 spreading Effects 0.000 claims description 11
- 239000012780 transparent material Substances 0.000 claims description 11
- 230000018044 dehydration Effects 0.000 claims description 10
- 238000006297 dehydration reaction Methods 0.000 claims description 10
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000007789 sealing Methods 0.000 claims description 10
- 238000003384 imaging method Methods 0.000 claims description 8
- 238000004043 dyeing Methods 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 5
- 108010039918 Polylysine Proteins 0.000 claims description 5
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims description 5
- 238000007667 floating Methods 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- 230000001788 irregular Effects 0.000 claims description 5
- 229920002866 paraformaldehyde Polymers 0.000 claims description 5
- 230000001575 pathological effect Effects 0.000 claims description 5
- 229920000656 polylysine Polymers 0.000 claims description 5
- 239000012192 staining solution Substances 0.000 claims description 5
- 238000009966 trimming Methods 0.000 claims description 5
- 235000016477 Taralea oppositifolia Nutrition 0.000 claims description 4
- 241001358109 Taralea oppositifolia Species 0.000 claims description 4
- 101150078127 MUSK gene Proteins 0.000 abstract description 55
- 238000011160 research Methods 0.000 abstract description 20
- 230000028327 secretion Effects 0.000 abstract description 16
- 230000007246 mechanism Effects 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 8
- 230000000877 morphologic effect Effects 0.000 abstract description 8
- 238000012800 visualization Methods 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 238000007794 visualization technique Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 43
- 230000000762 glandular Effects 0.000 description 7
- 239000006187 pill Substances 0.000 description 7
- 210000001732 sebaceous gland Anatomy 0.000 description 7
- 210000004204 blood vessel Anatomy 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 210000003205 muscle Anatomy 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 210000003484 anatomy Anatomy 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 210000003780 hair follicle Anatomy 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010000077 Abdominal mass Diseases 0.000 description 1
- 201000000736 Amenorrhea Diseases 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 241001090476 Castoreum Species 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 241000282375 Herpestidae Species 0.000 description 1
- 208000004044 Hypesthesia Diseases 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010068319 Oropharyngeal pain Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000003443 Unconsciousness Diseases 0.000 description 1
- 231100000540 amenorrhea Toxicity 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 208000010515 dystocia Diseases 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 208000034783 hypoesthesia Diseases 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 201000007227 lymph node tuberculosis Diseases 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000009112 niuhuang Substances 0.000 description 1
- 239000009528 niuhuang qingxin Substances 0.000 description 1
- 231100000862 numbness Toxicity 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 210000001034 respiratory center Anatomy 0.000 description 1
- 210000001104 scent gland Anatomy 0.000 description 1
- 230000035900 sweating Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 206010042772 syncope Diseases 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000000007 visual effect Effects 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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
Abstract
The invention provides a three-dimensional reconstruction method of forest musk gland tissues, and solves the technical problems that a forest musk secretion mechanism cannot be developed on the tissue and cell morphological structure level and further intensive research on the aspect of increasing musk yield in the prior art. It includes sampling, slicing, staining, scanning, three-dimensional reconstruction of the musk forest gland tissue, and the like. According to the three-dimensional reconstruction method of the musk forest gland tissue, the in-vitro visualization of the musk gland tissue is realized by tissue section staining based on the musk forest gland tissue, and a better application effect is obtained; the physiological structure of the musk forest gland tissue is researched by an in-vitro visualization method of the musk forest gland tissue, the three-dimensional in-vitro structure of the musk forest gland tissue is displayed for the first time, the morphological structure content of the musk forest gland tissue is enriched, and a morphological basis is provided for the research of deeply researching the musk forest secretion reaction and improving the musk yield.
Description
Technical Field
The invention relates to a three-dimensional reconstruction method of forest musk gland tissues.
Background
Musk is a famous and precious traditional Chinese medicine in China, has more than 2000 years of medicinal history, and is the first of four animal perfumes (musk, ambergris, civet and castoreum). It is warm in nature, pungent in flavor, and has effects of inducing resuscitation, dredging channels, relieving swelling and pain, etc., and can be widely used for treating fever unconsciousness, apoplexy, phlegm syncope, qi stagnation, coma, amenorrhea, abdominal mass, dystocia, stillborn fetus, thoracic obstruction, heart and abdominal pain, traumatic injury, numbness, carbuncle, scrofula, and sore throat. There are 295 more musks required in 2621 Chinese patent medicine prescriptions collected in China. Among 430 Chinese patent medicines produced by 11 animal medicinal materials, such as the commonly used Liushen pill, the Niuhuang Qingxin pill, the ginseng revival pill, the revival first-aid pill, the Anguan Niuhuang pill, the quick-acting heart-saving pill and the like, 186 musk are required, accounting for 43 percent of the total number, and the Musk pill belongs to a precious Chinese patent medicine. Pharmacological research proves that musk can promote secretion of glands, has the effects of sweating and diuresis, and has the effects of exciting central nervous system, respiratory center and heart, and also has the effects of resisting bacteria and diminishing inflammation. The medicine is used for treating coronary heart disease and angina pectoris and for cancer experiment. The Chinese and Western clinical experience proves that the musk also has curative effect on encephalitis B, syphilis, measles and other diseases.
The musk is prepared by curing the secretion liquid of the fragrant glands in the musk bag of a male forest, and is closely related to the physiological structure and the fragrance secretion mechanism of the musk bag. In the 80 s of the 20 th century, researchers have conducted relevant research on the musk secretion mechanism, but mainly focused on the anatomical structure and tissue structure of musk bags due to technical condition limitations. The research results enable people to have basic understanding on the macroscopic anatomical structure and the ultrastructure of the musk forest gland, and provide a reference theoretical basis for developing the musk forest secretion mechanism and improving musk yield on the aspect of tissue and cell morphological structure.
Due to the social and economic development reasons, in the 90 s of the world to the early century, due to the fact that investment in musk research is greatly reduced, a plurality of work is interrupted in succession, and the research on the fragrance secretion mechanism of the forest musk deer also goes into a dilemma. After 2010, forest musk secretion mechanism researches are added in Sichuan musk breeding research institute, Chongqing city medicine planting research institute, Beijing forestry university, Chinese people university, Shanxi province animal research institute, northwest agriculture and forestry science and technology university, Sichuan agriculture university and other disputes, but the research is hindered by the fact that the former research history is long, traceability cannot be performed, many HE dyeing pictures are blurred, and effective technical support cannot be provided for the modern secretion mechanism researches. In addition, the forest musk deer belongs to domestic secondary protection animals, the sampling difficulty always hinders the existing research, so that a plurality of researchers have unclear knowledge on the scent gland structure of the forest musk deer, and can not develop further deep research on the aspects of forest musk deer secretion mechanisms and musk deer yield improvement on the aspects of tissue and cell morphological structure.
Disclosure of Invention
The invention aims to provide a three-dimensional reconstruction method of a musk deer glandular tissue, which aims to solve the technical problem that the further intensive research on the aspects of developing a musk deer secretion mechanism and improving musk yield on the tissue and cell morphological structure level cannot be carried out in the prior art. The technical effects that can be produced by the preferred technical scheme in the technical schemes provided by the invention are described in detail in the following.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a three-dimensional reconstruction method of forest musk gland tissues, which comprises the following steps:
(1) sampling in vitro tissues: immediately taking the fragrant gland tissue of the male forest musk deer after the male forest musk deer naturally dies;
(2) fixing: immediately immersing the tonka tissue obtained in the step (1) into a 4% paraformaldehyde solution, and storing for more than 24h in a dark place to obtain a fixing material;
(3) and (3) dehydrating: taking out the fixing material in the step (2), trimming to flatten the cut surface, and dehydrating the fixing material trimmed and flattened by the cut surface in 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, 100% alcohol I for 30 minutes and 100% alcohol II for 30 minutes in sequence to obtain a dehydrated material;
(4) and (3) transparency: sequentially enabling the dehydrated material obtained in the step (3) to be transparent in a mixed solution of absolute ethyl alcohol and xylene for 15-30min, xylene I for 15-30min and xylene II for 15-30min to obtain a transparent material;
(5) wax dipping and embedding: sequentially dipping the transparent material obtained in the step (4) in paraffin I for 15-20min, dipping in paraffin II for 30min-1h and dipping in paraffin III for 30min-1h, and then embedding in an embedding machine to obtain a wax block;
(6) block repairing: cutting off the irregular part of the wax block obtained in the step (5) to ensure that the wax block has a regular shape and is convenient for slicing;
(7) slicing: placing the wax block trimmed in the step (6) on a pathological section machine to continuously slice for a plurality of times, wherein the slice thickness is 4 mu m;
(8) spreading: floating the slices obtained in the step (7) on a warm water surface at 38-40 ℃ for flattening, and then taking out the slices by using the anti-drop treated glass slide;
(9) baking and storing: putting the slices fished out in the step (8) into a 60 ℃ oven for baking, and storing at room temperature after baking is finished;
(10) paraffin section dewaxing to water: putting the slices treated in the step (9) into dimethylbenzene I for 20min, dimethylbenzene II for 20min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min and 75% alcohol for 5min in sequence, and finally washing with water;
(11) hematoxylin staining: placing the slices treated in the step (10) into hematoxylin staining solution for staining for 3-5min, and then washing with water; then putting differentiation liquid for differentiation, and then washing with water; finally returning blue liquid to blue for washing by running water;
(12) eosin staining: placing the slices treated in the step (11) into 85% alcohol for dehydration for 5min, placing the slices into 95% alcohol for dehydration for 5min, and then placing the slices into eosin dye solution for dyeing for 5 min;
(13) dewatering and sealing: putting the slices processed in the step (12) into absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, dimethyl I for 5min and xylene II for 5min in sequence, and sealing the slices with neutral gum;
(14) image acquisition: checking whether the slices processed in the step (13) are flat and have no cracks through a microscope one by one, carrying out image acquisition and analysis, and selecting the flat and non-crack slices to carry out the next step for image acquisition and analysis;
(15) scanning: placing all the slices screened in the step (14) under a microscope lens of a slice scanner in sequence to perform scanning imaging, seamlessly splicing each slice by a software control system of the slice scanner to generate a whole digital slice with a full view field, and obtaining one digital slice by each slice to obtain the digital slices;
(16) three-dimensional reconstruction: and (5) stacking all the digital slices scanned in the step (15) through 3DView software stack scanning pictures to form a three-dimensional stereogram.
Further, in the step (7), the number of the continuous slices is 150-1000.
Further, in the step (7), the number of the continuous slices is 450-850.
Further, in the step (4), the volume ratio of the anhydrous ethanol to the xylene in the mixed solution of the anhydrous ethanol and the xylene is 1: 1.
Further, in the step (8), the anti-shedding treatment is a polylysine treatment.
Based on the technical scheme, the embodiment of the invention can at least produce the following technical effects:
(1) according to the three-dimensional reconstruction method of the musk forest glandular tissue, the physiological structure of the musk forest glandular tissue is researched by an in-vitro visualization method of the musk forest glandular tissue, the three-dimensional in-vitro structure of the musk forest glandular tissue is displayed for the first time, the morphological structure content of the musk forest glandular tissue is enriched, and a morphological basis is provided for the deep research of musk forest secretion reaction and the research of musk yield improvement;
(2) the invention provides a three-dimensional reconstruction method of a musk forest gland tissue, which adopts more than 100 wax-block continuous slices, all the slices are subjected to HE dyeing, then all the slices are continuously scanned, pictures are scanned through a 3DView software stack to form a three-dimensional stereogram, relevant three-dimensional software (such as CaseViewer2.3 software) is utilized to carry out operations of cutting, rotating and the like to form the three-dimensional stereogram, so that the visualization of the musk gland tissue is rapidly realized, the in vitro visualized image of the musk forest gland tissue reproduced by the invention can clearly show the distribution of musk forest gland, muscle layers, sebaceous glands and blood vessels, and the trend and the corresponding relationship between the musk forest gland conduit and the blood vessels can be better shown; the exterior of the musk gland tissue is wrapped by the skin tissue which contains a large number of sebaceous glands and hair follicle tissues, so that the visual musk gland tissue is provided for researchers, and a foundation support is provided for promoting the research of the secretion mechanism of the forest musk deer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a 3D software imaging diagram (three-dimensional perspective view) in embodiment 1 of the present invention;
fig. 2 is a 3D software imaging diagram (three-dimensional perspective view) in embodiment 2 of the present invention;
fig. 3 is a 3D software imaging diagram (three-dimensional perspective view) in embodiment 3 of the present invention;
FIG. 4 is a sectional view of the tissue of the sachet in example 3 of the present invention;
FIG. 5 is a sebaceous gland secretion diagram in example 3 of the present invention;
FIG. 6 is a diagram showing the secretion of sebaceous glands at another site in example 3 of the present invention;
FIG. 7 is an enlarged view of a blood vessel tissue in example 3 of the present invention;
FIG. 8 is an enlarged view of another part of vascular tissue in example 3 of the present invention.
1. A muscle; 2. sebaceous glands; 3. a blood vessel; 4. and (5) fragrant glands.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
First, experimental equipment and experimental reagent specification
1. The experimental set-up is illustrated in table 1 below:
TABLE 1 Experimental Equipment
Table 2, description of the main experimental reagents, table 2 below:
TABLE 2 Main test reagents
| Name of reagent | Manufacturer of the product | Goods number |
| Anhydrous ethanol | SINOPHARM CHEMICAL REAGENT Co.,Ltd. | 100092683 |
| Xylene | SINOPHARM CHEMICAL REAGENT Co.,Ltd. | 10023418 |
| HE dye liquor | servicebio | G1005 |
| Differentiation liquid | servicebio | G1005-3 |
| Blue-returning liquid | servicebio | G1005-4 |
| Neutral gum | SINOPHARM CHEMICAL REAGENT Co.,Ltd. | 10004160 |
Second, embodiment:
example 1:
the invention provides a three-dimensional reconstruction method of forest musk gland tissues, which comprises the following steps:
(1) sampling in vitro tissues: immediately taking the fragrant gland tissue of the male forest musk deer after the male forest musk deer naturally dies;
(2) fixing: immediately immersing the tonka tissue obtained in the step (1) into a 4% paraformaldehyde solution, and storing for 24h in a dark place to obtain a fixing material;
(3) and (3) dehydrating: dehydrating by using a dehydrator, taking out the fixing material in the step (2), trimming to flatten the cut surface, dehydrating the fixing material trimmed and flattened by the cut surface in 75% alcohol for 4 hours, dehydrating in 85% alcohol for 2 hours, dehydrating in 90% alcohol for 2 hours, dehydrating in 95% alcohol for 1 hour, dehydrating in 100% alcohol I for 30min, and dehydrating in 100% alcohol II for 30min to obtain a dehydrated material;
(4) and (3) transparency: sequentially enabling the dehydrated material obtained in the step (3) to be transparent in a mixed solution of absolute ethyl alcohol and xylene for 30min, transparent in xylene I for 30min and transparent in xylene II for 30min to obtain a transparent material;
in the mixed solution of the anhydrous ethanol and the xylene, the volume ratio of the anhydrous ethanol to the xylene is 1: 1;
(5) wax dipping and embedding: sequentially soaking the transparent material obtained in the step (4) in paraffin I for 20min, paraffin II for 30min and paraffin III for 30min, and then embedding the transparent material in an embedding machine to obtain a wax block;
(6) block repairing: cutting off the irregular part of the wax block obtained in the step (5) to ensure that the wax block has a regular shape and is convenient for slicing;
(7) slicing: placing the wax block trimmed in the step (6) on a pathological section machine to continuously slice for a plurality of times, wherein the slice thickness is 4 mu m;
in the step (7), the number of the continuous slices is 150;
(8) spreading: spreading the slices by using a tissue spreading machine, floating the slices obtained in the step (7) on a warm water surface at 40 ℃, flattening the slices, and then fishing out the slices by using the anti-drop treated glass slide;
the anti-dropping treatment is polylysine treatment;
(9) baking and storing: putting the slices fished out in the step (8) into a 60 ℃ oven for baking, and storing at room temperature after baking is finished;
(10) paraffin section dewaxing to water: putting the slices treated in the step (9) into dimethylbenzene I for 20min, dimethylbenzene II for 20min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min and 75% alcohol for 5min in sequence, and finally washing with water;
(11) hematoxylin staining: placing the slices treated in the step (10) into hematoxylin staining solution for staining for 5min, and then washing with water; then putting differentiation liquid for differentiation, and then washing with water; finally returning blue liquid to blue for washing by running water;
(12) eosin staining: placing the slices treated in the step (11) into 85% alcohol for dehydration for 5min, placing the slices into 95% alcohol for dehydration for 5min, and then placing the slices into eosin dye solution for dyeing for 5 min;
(13) dewatering and sealing: putting the slices processed in the step (12) into absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, dimethyl I for 5min and xylene II for 5min in sequence, and sealing the slices with neutral gum;
(14) image acquisition: examining whether the slices processed in the step (13) are flat and crack-free through a microscope (adopting an upright optical microscope and a digital microscopic imaging system) one by one, carrying out image acquisition and analysis, and selecting the flat and crack-free slices to carry out the next step;
(15) scanning: placing all the slices screened in the step (14) under a microscope lens of a slice scanner in sequence to perform scanning imaging, seamlessly splicing each slice by a software control system of the slice scanner to generate a whole digital slice with a full view field, and obtaining one digital slice by each slice to obtain the digital slices;
(16) three-dimensional reconstruction: stacking all the digital slices scanned in the step (15) through a 3DView software stack scanning picture to form a three-dimensional stereogram; the 3d view software is a caseviewer2.3 software, and can be used for performing operations such as cutting and rotating to form a three-dimensional stereo map.
Example 2:
the invention provides a three-dimensional reconstruction method of forest musk gland tissues, which comprises the following steps:
(1) sampling in vitro tissues: immediately taking the fragrant gland tissue of the male forest musk deer after the male forest musk deer naturally dies;
(2) fixing: immediately immersing the tonka tissue obtained in the step (1) into a 4% paraformaldehyde solution, and storing for 48 hours in a dark place to obtain a fixing material;
(3) and (3) dehydrating: dehydrating by using a dehydrator, taking out the fixing material in the step (2), trimming to flatten the cut surface, dehydrating the fixing material trimmed and flattened by the cut surface in 75% alcohol for 4 hours, dehydrating in 85% alcohol for 2 hours, dehydrating in 90% alcohol for 2 hours, dehydrating in 95% alcohol for 1 hour, dehydrating in 100% alcohol I for 30min, and dehydrating in 100% alcohol II for 30min to obtain a dehydrated material;
(4) and (3) transparency: sequentially enabling the dehydrated material obtained in the step (3) to be transparent in a mixed solution of absolute ethyl alcohol and xylene for 15min, transparent in xylene I for 15min and transparent in xylene II for 15min to obtain a transparent material;
in the mixed solution of the anhydrous ethanol and the xylene, the volume ratio of the anhydrous ethanol to the xylene is 1: 1;
(5) wax dipping and embedding: sequentially dipping the transparent material obtained in the step (4) in paraffin I for 15min, dipping in paraffin II for 45min and dipping in paraffin III for 45min, and then embedding by an embedding machine to obtain a wax block;
(6) block repairing: cutting off the irregular part of the wax block obtained in the step (5) to ensure that the wax block has a regular shape and is convenient for slicing;
(7) slicing: placing the wax block trimmed in the step (6) on a pathological section machine to continuously slice for a plurality of times, wherein the slice thickness is 4 mu m;
in the step (7), the number of the continuous slices is 1000;
(8) spreading: spreading the slices by using a tissue spreading machine, floating the slices obtained in the step (7) on a warm water surface at 39 ℃, flattening the slices, and then fishing out the slices by using the anti-drop treated glass slide;
the anti-dropping treatment is polylysine treatment;
(9) baking and storing: putting the slices fished out in the step (8) into a 60 ℃ oven for baking, and storing at room temperature after baking is finished;
(10) paraffin section dewaxing to water: putting the slices treated in the step (9) into dimethylbenzene I for 20min, dimethylbenzene II for 20min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min and 75% alcohol for 5min in sequence, and finally washing with water;
(11) hematoxylin staining: placing the slices treated in the step (10) into hematoxylin staining solution for staining for 3min, and then washing with water; then putting differentiation liquid for differentiation, and then washing with water; finally returning blue liquid to blue for washing by running water;
(12) eosin staining: placing the slices treated in the step (11) into 85% alcohol for dehydration for 5min, placing the slices into 95% alcohol for dehydration for 5min, and then placing the slices into eosin dye solution for dyeing for 5 min;
(13) dewatering and sealing: putting the slices processed in the step (12) into absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, dimethyl I for 5min and xylene II for 5min in sequence, and sealing the slices with neutral gum;
(14) image acquisition: examining whether the slices processed in the step (13) are flat and crack-free through a microscope (adopting an upright optical microscope and a digital microscopic imaging system) one by one, carrying out image acquisition and analysis, and selecting the flat and crack-free slices to carry out the next step;
(15) scanning: placing all the slices screened in the step (14) under a microscope lens of a slice scanner in sequence to perform scanning imaging, seamlessly splicing each slice by a software control system of the slice scanner to generate a whole digital slice with a full view field, and obtaining one digital slice by each slice to obtain the digital slices;
(16) three-dimensional reconstruction: stacking all the digital slices scanned in the step (15) through a 3DView software stack scanning picture to form a three-dimensional stereogram; the 3d view software is a caseviewer2.3 software, and can be used for performing operations such as cutting and rotating to form a three-dimensional stereo map.
Example 3:
a three-dimensional reconstruction method of forest musk gland tissues comprises the following steps:
(1) sampling in vitro tissues: immediately taking the fragrant gland tissue of the male forest musk deer after the male forest musk deer naturally dies;
(2) fixing: immediately immersing the glandular tissue obtained in the step (1) into a 4% paraformaldehyde solution, and keeping the mixture in the dark for 36 hours to obtain a fixing material;
(3) and (3) dehydrating: dehydrating by using a dehydrator, taking out the fixing material in the step (2), trimming to flatten the cut surface, dehydrating the fixing material trimmed and flattened by the cut surface in 75% alcohol for 4 hours, dehydrating in 85% alcohol for 2 hours, dehydrating in 90% alcohol for 2 hours, dehydrating in 95% alcohol for 1 hour, dehydrating in 100% alcohol I for 30min, and dehydrating in 100% alcohol II for 30min to obtain a dehydrated material;
(4) and (3) transparency: sequentially enabling the dehydrated material obtained in the step (3) to be transparent in a mixed solution of absolute ethyl alcohol and xylene for 22min, xylene I for 22min and xylene II for 22min to obtain a transparent material;
in the mixed solution of the anhydrous ethanol and the dimethylbenzene, the volume ratio of the anhydrous ethanol to the dimethylbenzene is 1:1
(5) Wax dipping and embedding: sequentially soaking the transparent material obtained in the step (4) in paraffin I for 18min, soaking in paraffin II for 1h and soaking in paraffin III for 1h, and then embedding by using an embedding machine to obtain a wax block;
(6) block repairing: cutting off the irregular part of the wax block obtained in the step (5) to ensure that the wax block has a regular shape and is convenient for slicing;
(7) slicing: placing the wax block trimmed in the step (6) on a pathological section machine to continuously slice for a plurality of times, wherein the slice thickness is 4 mu m;
the number of the continuous slices is 750;
(8) spreading: spreading the slices by using a tissue spreading machine, floating the slices obtained in the step (7) on a warm water surface at 40 ℃, flattening the slices, and then fishing out the slices by using the anti-drop treated glass slide;
the anti-dropping treatment is polylysine treatment;
(9) baking and storing: putting the slices fished out in the step (8) into a 60 ℃ oven for baking, and storing at room temperature after baking is finished;
(10) paraffin section dewaxing to water: putting the slices treated in the step (9) into dimethylbenzene I for 20min, dimethylbenzene II for 20min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min and 75% alcohol for 5min in sequence, and finally washing with water;
(11) hematoxylin staining: placing the slices treated in the step (10) into hematoxylin staining solution for staining for 4min, and then washing with water; then putting differentiation liquid for differentiation, and then washing with water; finally returning blue liquid to blue for washing by running water;
(12) eosin staining: placing the slices treated in the step (11) into 85% alcohol for dehydration for 5min, placing the slices into 95% alcohol for dehydration for 5min, and then placing the slices into eosin dye solution for dyeing for 5 min;
(13) dewatering and sealing: putting the slices processed in the step (12) into absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, dimethyl I for 5min and xylene II for 5min in sequence, and sealing the slices with neutral gum;
(14) image acquisition: examining whether the slices processed in the step (13) are flat and crack-free through a microscope (adopting an upright optical microscope and a digital microscopic imaging system) one by one, carrying out image acquisition and analysis, and selecting the flat and crack-free slices to carry out the next step;
(15) scanning: placing all the slices screened in the step (14) under a microscope lens of a slice scanner in sequence to perform scanning imaging, seamlessly splicing each slice by a software control system of the slice scanner to generate a whole digital slice with a full view field, and obtaining one digital slice by each slice to obtain the digital slices;
(16) three-dimensional reconstruction: stacking all the digital slices scanned in the step (15) through a 3DView software stack scanning picture to form a three-dimensional stereogram; the 3d view software is a caseviewer2.3 software, and can be used for performing operations such as cutting and rotating to form a three-dimensional stereo map.
Example 4:
in this embodiment, in the step (7), the number of the continuous slices is 450; the rest is the same as example 3.
Example 5:
in this embodiment, in the step (7), the number of the continuous slices is 850; the rest is the same as example 3.
As shown in fig. 1-8:
the in vitro visualization image of the musk forest gland tissue reproduced by the method can clearly show the distribution of musk forest gland, muscle layers, sebaceous glands and blood vessels, and can better show the trend of the musk forest gland conduit and the corresponding relation between the musk forest gland conduit and the blood vessels; the exterior of the balsamic gland tissue is wrapped by the skin tissue which contains a large amount of sebaceous gland and hair follicle tissues, which can be seen from the three-dimensional reconstructed stereo image.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention.
Claims (5)
1. A three-dimensional reconstruction method of forest musk gland tissues is characterized by comprising the following steps: the method comprises the following steps:
(1) sampling in vitro tissues: immediately taking the fragrant gland tissue of the male forest musk deer after the male forest musk deer naturally dies;
(2) fixing: immediately immersing the tonka tissue obtained in the step (1) into a 4% paraformaldehyde solution, and storing for more than 24h in a dark place to obtain a fixing material;
(3) and (3) dehydrating: taking out the fixing material in the step (2), trimming to flatten the cut surface, and dehydrating the fixing material trimmed and flattened by the cut surface in 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, 100% alcohol I for 30 minutes and 100% alcohol II for 30 minutes in sequence to obtain a dehydrated material;
(4) and (3) transparency: sequentially enabling the dehydrated material obtained in the step (3) to be transparent in a mixed solution of absolute ethyl alcohol and xylene for 15-30min, xylene I for 15-30min and xylene II for 15-30min to obtain a transparent material;
(5) wax dipping and embedding: sequentially dipping the transparent material obtained in the step (4) in paraffin I for 15-20min, dipping in paraffin II for 30min-1h and dipping in paraffin III for 30min-1h, and then embedding in an embedding machine to obtain a wax block;
(6) block repairing: cutting off the irregular part of the wax block obtained in the step (5) to ensure that the wax block has a regular shape and is convenient for slicing;
(7) slicing: placing the wax block trimmed in the step (6) on a pathological section machine to continuously slice for a plurality of times, wherein the slice thickness is 4 mu m;
(8) spreading: floating the slices obtained in the step (7) on a warm water surface at 38-40 ℃ for flattening, and then taking out the slices by using the anti-drop treated glass slide;
(9) baking and storing: putting the slices fished out in the step (8) into a 60 ℃ oven for baking, and storing at room temperature after baking is finished;
(10) paraffin section dewaxing to water: putting the slices treated in the step (9) into dimethylbenzene I for 20min, dimethylbenzene II for 20min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min and 75% alcohol for 5min in sequence, and finally washing with water;
(11) hematoxylin staining: placing the slices treated in the step (10) into hematoxylin staining solution for staining for 3-5min, and then washing with water; then putting differentiation liquid for differentiation, and then washing with water; finally returning blue liquid to blue for washing by running water;
(12) eosin staining: placing the slices treated in the step (11) into 85% alcohol for dehydration for 5min, placing the slices into 95% alcohol for dehydration for 5min, and then placing the slices into eosin dye solution for dyeing for 5 min;
(13) dewatering and sealing: putting the slices processed in the step (12) into absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, absolute ethyl alcohol III for 5min, dimethyl I for 5min and xylene II for 5min in sequence, and sealing the slices with neutral gum;
(14) image acquisition: checking whether the slices processed in the step (13) are flat and have no cracks through a microscope one by one, carrying out image acquisition and analysis, and selecting the flat and non-crack slices to carry out the next step;
(15) scanning: placing all the slices screened in the step (14) under a microscope lens of a slice scanner in sequence to perform scanning imaging, seamlessly splicing each slice by a software control system of the slice scanner to generate a whole digital slice with a full view field, and obtaining one digital slice by each slice to obtain the digital slices;
(16) three-dimensional reconstruction: and (5) stacking all the digital slices scanned in the step (15) through 3DView software stack scanning pictures to form a three-dimensional stereogram.
2. The method of three-dimensional reconstruction of a musk forest gland tissue according to claim 1, wherein: in the step (7), the number of the continuous slices is 150-1000.
3. The method of three-dimensional reconstruction of a musk forest gland tissue according to claim 1, wherein: in the step (7), the number of the continuous slices is 450 and 850.
4. The method of three-dimensional reconstruction of a musk forest gland tissue according to claim 1, wherein: in the step (4), the volume ratio of the absolute ethyl alcohol to the xylene in the mixed solution of the absolute ethyl alcohol and the xylene is 1: 1.
5. The method of three-dimensional reconstruction of a musk forest gland tissue according to claim 1, wherein: in the step (8), the anti-dropping treatment is polylysine treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011371039.6A CN112393962A (en) | 2020-11-30 | 2020-11-30 | Three-dimensional reconstruction method of forest musk gland tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011371039.6A CN112393962A (en) | 2020-11-30 | 2020-11-30 | Three-dimensional reconstruction method of forest musk gland tissue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112393962A true CN112393962A (en) | 2021-02-23 |
Family
ID=74604713
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011371039.6A Pending CN112393962A (en) | 2020-11-30 | 2020-11-30 | Three-dimensional reconstruction method of forest musk gland tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112393962A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115082529A (en) * | 2022-06-30 | 2022-09-20 | 华东师范大学 | System and method for collecting and analyzing multi-dimensional information of gross tissue |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915693A (en) * | 2010-07-07 | 2010-12-15 | 新疆医科大学 | Human embryonic trigeminus based three-dimensional reconstruction method by using histotomy staining |
| US20170108414A1 (en) * | 2015-10-20 | 2017-04-20 | University Of Washington | High-resolution three-dimensional imaging of mammalian hearts |
| CN109859304A (en) * | 2018-11-16 | 2019-06-07 | 华中科技大学同济医学院附属同济医院 | Three-dimensional printing technology establishes the application in three-dimensional structure digital model in corneal limbal tissue in vitro |
| CN109884019A (en) * | 2019-03-25 | 2019-06-14 | 大连大学 | A kind of three-dimension curved surface reconstructing method that biomembrane is applicable |
| CN111257090A (en) * | 2019-12-18 | 2020-06-09 | 武汉沃亿生物有限公司 | Dil ultrasonic staining-based biological tissue sample three-dimensional imaging method |
-
2020
- 2020-11-30 CN CN202011371039.6A patent/CN112393962A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915693A (en) * | 2010-07-07 | 2010-12-15 | 新疆医科大学 | Human embryonic trigeminus based three-dimensional reconstruction method by using histotomy staining |
| US20170108414A1 (en) * | 2015-10-20 | 2017-04-20 | University Of Washington | High-resolution three-dimensional imaging of mammalian hearts |
| CN109859304A (en) * | 2018-11-16 | 2019-06-07 | 华中科技大学同济医学院附属同济医院 | Three-dimensional printing technology establishes the application in three-dimensional structure digital model in corneal limbal tissue in vitro |
| CN109884019A (en) * | 2019-03-25 | 2019-06-14 | 大连大学 | A kind of three-dimension curved surface reconstructing method that biomembrane is applicable |
| CN111257090A (en) * | 2019-12-18 | 2020-06-09 | 武汉沃亿生物有限公司 | Dil ultrasonic staining-based biological tissue sample three-dimensional imaging method |
Non-Patent Citations (1)
| Title |
|---|
| 芮菊生等: "麝香腺分泌周期中组织学及组织化学的初步研究", 《南通医学院学报》, vol. 8, no. 4, pages 26 - 5 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115082529A (en) * | 2022-06-30 | 2022-09-20 | 华东师范大学 | System and method for collecting and analyzing multi-dimensional information of gross tissue |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106420460A (en) | Ginseng water energizing cream, preparation method thereof and mask containing ginseng water energizing cream | |
| CN103976908B (en) | A kind ofly be rich in the composition of plant extracts of plant polypeptide and the application in cosmetics thereof | |
| CN105395440B (en) | A kind of peach gum facial mask and its preparation method and application | |
| CN106137916A (en) | The cosmetic composition of a kind of removing acnes and controlling oil whitening and application thereof | |
| Giuffra et al. | Embalming methods and plants in Renaissance Italy: two artificial mummies from Siena (central Italy) | |
| CN102836114A (en) | Skin-moistening emulsion | |
| CN112393962A (en) | Three-dimensional reconstruction method of forest musk gland tissue | |
| CN106420459B (en) | A kind of ginseng water Essence living and preparation method thereof and the facial mask comprising it | |
| CN107582729A (en) | A kind of preparation method and applications of belladonna extract | |
| CN104491003B (en) | Add taste Yupingfeng gelling agent and its preparation method and application | |
| CN1301745C (en) | Chinese medicinal liquid for treating alopecia and its production method | |
| CN108670910A (en) | A kind of profit hair composition and preparation method thereof | |
| CN104095883A (en) | Coral extract, its extraction method and use, and coral extract health product | |
| CN108272533A (en) | The skin modeling method in skin wound region | |
| CN104800269A (en) | Traditional Chinese medicine external-use cream used for treating psoriasis and preparation method thereof | |
| CN108670933B (en) | Skin care composition with moisturizing, whitening and anti-aging functions and application thereof | |
| CN104593208A (en) | Living body male silkworm moth liquor and preparation method thereof | |
| CN102764226B (en) | Eyebrow nourishing liquid, and preparation method and application thereof | |
| CN110558280A (en) | Preparation method of liver cancer animal model | |
| CN114848569A (en) | Formula and preparation method of hair loss prevention and hair nourishing product | |
| CN114949029A (en) | Hypericum perforatum extract for promoting wound healing and preparation method thereof | |
| CN113925811A (en) | Biological energy breast enlargement cream for instruments and preparation method | |
| CN106038423B (en) | A kind of anti-acne, except scar gel and preparation method thereof | |
| CN112237600B (en) | Application of compound zanthoxylum bungeanum gel in preparation of product for preventing and/or treating chilblain | |
| CN108514530A (en) | Anti-wrinkle cosmetic cosmetics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |