CN100567943C - A kind of section statining method of skeletal muscle band - Google Patents
A kind of section statining method of skeletal muscle band Download PDFInfo
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- CN100567943C CN100567943C CN 200710051996 CN200710051996A CN100567943C CN 100567943 C CN100567943 C CN 100567943C CN 200710051996 CN200710051996 CN 200710051996 CN 200710051996 A CN200710051996 A CN 200710051996A CN 100567943 C CN100567943 C CN 100567943C
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- alcohol
- skeletal muscle
- wax
- band
- dimethylbenzene
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Abstract
The present invention relates to a kind of section statining method of skeletal muscle band, get skeletal muscle and put into immobile liquid, fixedly after 4-6 hour skeletal muscle is cut into the piece of tissue of 3-5 millimeter, and then put into immobile liquid fixedly 8-18 hour; Take out the flushing of skeletal muscle tissue piece flowing water and be placed on potassinm permanganate haematine dye liquor dyeing 10-12 days, use 70% alcohol-pickled 15-30 minute then, the flowing water flushing respectively lasts 2 hours from 70% alcohol, 80% alcohol, each liquid of 90% alcohol step by step, puts into eosin stain dyeing again; The skeletal muscle tissue piece is gone into 95% alcohol, 100% dehydration of alcohol each 2 hours; Soak 2 times going into soft wax after the skeletal muscle tissue piece immersion dimethylbenzene transparent processing; Hard wax soaks 2 times; The wax stone that obtains after the paraffin embedding is cut into the wax band of 4-6 micron thickness with microtome; The wax disk(-sc) that wax band branch is cut into the 0.8-1 centimeter length is attached on the microslide; Volatilize fully with being heated to dimethylbenzene after the dimethylbenzene dewaxing behind the paster, treat to use the neutral gum mounting after the paster drying.The present invention can clear display the skeletal muscle band.
Description
Technical field
The present invention relates to a kind of section statining method of skeletal muscle band, belong to the organic section making technical field.
Background technology
Skeletal muscle tissue section occupies critical role in the teaching process of histology, pathology, biology etc., demand is big in teaching process, is a very complicated loaded down with trivial details technology, and the requirement of technology is strict.
Colouring method is one step of key that shows the skeletal muscle band in the film-making process.The three major types colouring method that the colouring method that is used for skeletal muscle at present mainly contains under the light microscopic, fluorescent microscope reaches electron microscopic observation down.Be used for the colouring method observed under fluorescent microscope and the Electronic Speculum the effect form of visualize tissue cell clearly, and colouring method is many, as TUNEL etc. according to himself.But the price comparison costliness of the colouring method film-making of this two big class cost, and the hardware facility that requires does not just reach this requirement than higher as middle school, institution of higher learning at all.
Be applicable to that optical microscope and present most popular colouring method are exactly HE dyeing.But the very loaded down with trivial details (Bing Lujun of the workflow of HE stained preparation, Kau Ying-mau, the Guo Yu shepherd's purse, Xiaoli Zhang improves the technology [J] of histology experiment teaching paraffin section quality. and the laboratory is explored, 2004,23 (12): 152-153.), rip cutting can show the skeletal muscle band, but band is more tiny, and of low quality.Also need therefrom screening for producing in enormous quantities, just more increased workload.
Summary of the invention
Problem to be solved by this invention provides a kind of section statining method of skeletal muscle band, and the skeletal muscle band that the skeletal muscle section of handling through the present invention can clear display is fit to produce in enormous quantities.
Technical scheme provided by the invention is: a kind of section statining method of skeletal muscle band, and get skeletal muscle and put into immobile liquid, fixedly after 4-6 hour skeletal muscle is cut into the piece of tissue of 3-5 millimeter, and then put into immobile liquid fixedly 8-18 hour; Take out the flushing of skeletal muscle tissue piece flowing water and be placed on potassinm permanganate haematine dye liquor dyeing 10-12 days, use 70% alcohol-pickled (being color separation) 15-30 minute then, flowing water flushing 20-30 hour, respectively last 2 hours from 70% alcohol, 80% alcohol, each liquid of 90% alcohol step by step, put into eosin stain dyeing 2-3 days again; The skeletal muscle tissue piece is gone into 95% alcohol, 100% dehydration of alcohol each 2 hours; Skeletal muscle tissue piece immersion dimethylbenzene transparent processing is gone into soft wax after 14-16 hour soak each 40-60 minute 2 times; Hard wax soaks 2 times, each 40-60 minute; The wax stone that obtains after the paraffin embedding is cut into the wax band of 4-6 micron thickness with microtome; The wax disk(-sc) that wax band branch is cut into the 0.8-1 centimeter length is attached on the microslide; Be heated to dimethylbenzene with the dimethylbenzene dewaxing after 20-30 minute behind the paster and volatilize fully, treat to use the neutral gum mounting after the paster drying.
Said fixing liquid is the nitric acid immobile liquid of being made up of anhydrous alcohol, distilled water and nitric acid, and wherein the consumption volume ratio of anhydrous alcohol, distilled water and nitric acid is 100: 16: 4.
Above-mentioned potassinm permanganate brazilwood extract dyeing liquid is made up of haematine, potassium permanganate, aluminium potassium sulfate and distilled water, and wherein the consumption weight ratio of haematine, potassium permanganate, aluminium potassium sulfate and distilled water is 0.11: 0.102: 5: 100.
Above-mentioned Yihong dyeing liquor is made up of 95% alcohol and Yihong, and wherein every 100ml alcohol contains 0.25g Yihong.
The skeletal muscle section of handling through the present invention can clear display the skeletal muscle band, the success ratio height is fit to produce in enormous quantities.
Description of drawings
Fig. 1 handles the microphoto of the skeletal muscle section that obtains for the embodiment of the invention 1.
Fig. 2 handles the microphoto of the skeletal muscle section that obtains for the embodiment of the invention 2.
Fig. 3 handles the microphoto of the skeletal muscle section that obtains for the embodiment of the invention 3.
Embodiment
Method for making is as follows:
1. the preparation of reagent
The immobile liquid preparation
100% alcohol 100ml, distilled water 16ml and nitric acid 4ml mixing are fixed liquid.
The preparation of haematine dye liquor
Haematine 0.11g, potassium permanganate 0.102g, aluminium potassium sulfate 5g and distilled water 100ml mixing are obtained the haematine dye liquor.
The preparation of eosin stain
95% alcohol 100m and Yihong 0.25g mixing are obtained eosin stain.
2. draw materials and fix: the animal that lives is killed (perhaps just dead animal), get the skeletal muscle at thigh place rapidly, put into immobile liquid immediately, number is 12-24 hour (take out the piece of tissue that skeletal muscle is cut into the 3-5 millimeter after fixing 4 hours, it is fixing to put into immobile liquid then) in the time of fixedly.
3. flushing: flowing water flushing 24 hours.
4. dyeing: skeletal muscle is gone into potassinm permanganate haematine dye liquor, and piece dyed 10-12 days.Use 70% alcohol color separation 15-30 minute then, flowing water flushing 20-30 hour lasts 2 hours from 70% alcohol, 80% alcohol, each liquid of 90% alcohol step by step, goes into Yihong dyeing 2-3 days then.
5. dehydration: skeletal muscle is gone into 95% alcohol, 100% dehydration of alcohol each 2 hours.
6. transparent: skeletal muscle was gone into the transparent 14-16 of dimethylbenzene hour.
7. waxdip and embedding: skeletal muscle is gone into soft wax and is soaked 2 times, each 40-60 minute; Hard wax soaks 2 times, each 40-60 minute; The routine paraffin wax embedding.
8. section: the wax band that wax stone is cut into the 4-6 micron thickness with microtome.
9. paster and roasting sheet: the wax disk(-sc) that the wax band is cut into the 0.8-1 centimeter length, dip in little glass rod and to get albumen glycerine, drip on a clean microslide, smear evenly with finger, and add several distilled water on microslide, put down gently with pincet gripping wax disk(-sc) and again microslide is being placed on the water surface of microslide on the exhibition sheet platform (temperature is about 45 degrees centigrade).Treat wax disk(-sc) be heated fully flatten after, incline and unload redundant moisture on the slide, with dissecting needle set right in the wax disk(-sc) position, be placed on then in the constant temperature oven about 50 degrees centigrade and dry.
10. dewaxing: section is through dimethylbenzene dewaxing 2 times, each 20-30min.
11. mounting: the section that will take out from dimethylbenzene is placed on spirit lamp and is heated to dimethylbenzene and volatilizees fully, uses the neutral gum mounting after the slice drying.
Just can examine under a microscope then.
Embodiment 1: get bullfrog skeletal muscle and put into immobile liquid, take out skeletal muscle and be cut into 5 millimeters piece of tissue after fixing 4 hours, put into immobile liquid again and fix 20 hours.The flowing water flushing is placed on potassinm permanganate haematine dye liquor dyeing 12 days, uses 70% alcohol color separation 30 minutes then, and flowing water flushing 24 hours respectively lasts 2 hours from 70% alcohol, 80% alcohol, each liquid of 90% alcohol step by step, goes into eosin stain dyeing 3 days then.The skeletal muscle tissue piece was gone into 95% alcohol, 100% dehydration of alcohol each 2 hours, and go into dimethylbenzene and go into soft wax after transparent 16 hours and soak 2 times, each about 60 minutes, totally 2 hours; Hard wax soaks 2 times, and each about 60 minutes, totally 2 hours.The routine paraffin wax embedding.Wax stone is cut into the wax band of 5 micron thickness with microtome.The wax disk(-sc) that wax band branch is cut into the 0.8-1 centimeter length is attached on the microslide, paster is after dimethylbenzene dewaxing 2 times, each 25min, the paster that takes out from dimethylbenzene is placed on spirit lamp and is heated to dimethylbenzene and volatilizees fully, treat to use the neutral gum mounting after the paster drying, microscopically (referring to Fig. 1) observations shows, the skeletal muscle band that the skeletal muscle section of handling through the present invention can clear display.
Embodiment 2: get bullfrog skeletal muscle and put into immobile liquid, take out skeletal muscle and tissue is cut into 3 millimeters bulk after fixing 6 hours, put into immobile liquid again and fix 6 hours.The flowing water flushing is placed on potassinm permanganate haematine dye liquor dyeing 10 days, uses 70% alcohol color separation 15 minutes then, and flowing water flushing 20 hours respectively lasts 2 hours from 70% alcohol, 80% alcohol, each liquid of 90% alcohol step by step, goes into eosin stain dyeing 2 days then.The skeletal muscle tissue piece was gone into 95% alcohol, 100% dehydration of alcohol each 2 hours, went into dimethylbenzene and went into soft wax after transparent 14 hours and soak each 45 minutes 2 times; Hard wax soaks 2 times, each 45 minutes.The routine paraffin wax embedding.Wax stone is cut into the wax band of 6 micron thickness with microtome.The wax disk(-sc) that wax band branch is cut into the 0.8-1 centimeter length is attached on the microslide, paster is after dimethylbenzene dewaxing 2 times, each 20min, the paster that takes out from dimethylbenzene is placed on spirit lamp and is heated to dimethylbenzene and volatilizees fully, treat to use the neutral gum mounting after the paster drying, microscopically (referring to Fig. 2) observations shows, the skeletal muscle band that the skeletal muscle section of handling through the present invention can clear display.
Embodiment 3: get bullfrog skeletal muscle and put into immobile liquid, take out skeletal muscle after fixing 5 hours to be cut into 4 millimeters piece of tissue, put into immobile liquid again and fix 15 hours.The flowing water flushing is placed on potassinm permanganate haematine dye liquor dyeing 10 days, uses 70% alcohol color separation 20 minutes then, and flowing water flushing 25 hours respectively lasts 2 hours from 70% alcohol, 80% alcohol, each liquid of 90% alcohol step by step, goes into eosin stain dyeing 3 days then.The skeletal muscle tissue piece was gone into 95% alcohol, 100% dehydration of alcohol each 2 hours, went into dimethylbenzene and went into soft wax after transparent 15 hours and soak each 40 minutes 2 times; Hard wax soaks 2 times, each 60 minutes.The routine paraffin wax embedding.Wax stone is cut into the wax band of 4 micron thickness with microtome.The wax disk(-sc) that wax band branch is cut into the 0.8-1 centimeter length is attached on the microslide, paster is after dimethylbenzene dewaxing 2 times, each 30min, the paster that takes out from dimethylbenzene is placed on spirit lamp and is heated to dimethylbenzene and volatilizees fully, treat to use the neutral gum mounting after the paster drying, microscopically (referring to Fig. 3) observations shows, the skeletal muscle band that the skeletal muscle section of handling through the present invention can clear display.
Claims (1)
1. the section statining method of a skeletal muscle band is got skeletal muscle and is put into immobile liquid, fixedly after 4-6 hour skeletal muscle is cut into the piece of tissue of 3-5 millimeter, and then puts into immobile liquid fixedly 8-18 hour; Take out the flushing of skeletal muscle tissue piece flowing water and be placed on potassinm permanganate haematine dye liquor dyeing 10-12 days, use 70% alcohol-pickled color separation 15-30 minute then, flowing water flushing 20-30 hour, respectively last 2 hours from 70% alcohol, 80% alcohol, each liquid of 90% alcohol step by step, put into eosin stain dyeing 2-3 days again; The skeletal muscle tissue piece is gone into 95% alcohol, 100% dehydration of alcohol each 2 hours; Skeletal muscle tissue piece immersion dimethylbenzene transparent processing is gone into soft wax after 14-16 hour soak each 40-60 minute 2 times; Hard wax soaks 2 times, each 40-60 minute; The wax stone that obtains after the paraffin embedding is cut into the wax band of 4-6 micron thickness with microtome; The wax disk(-sc) that wax band branch is cut into the 0.8-1 centimeter length is attached on the microslide; Paster is placed in the constant temperature oven about 50 degrees centigrade and dries, again with dimethylbenzene dewaxing 2 times, and each 20-30 minute, be heated to dimethylbenzene then and volatilize fully, treat to use the neutral gum mounting after the paster drying; Described immobile liquid is the nitric acid immobile liquid of being made up of anhydrous alcohol, distilled water and nitric acid, and wherein the consumption volume ratio of anhydrous alcohol, distilled water and nitric acid is 100: 16: 4; Described potassinm permanganate haematine dye liquor is made up of haematine, potassium permanganate, aluminium potassium sulfate and distilled water, and wherein the consumption weight ratio of haematine, potassium permanganate, aluminium potassium sulfate and distilled water is 0.11: 0.102: 5: 100; Described eosin stain is made up of 95% alcohol and Yihong, and wherein every 100ml alcohol contains 0.25g Yihong.
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| CN 200710051996 CN100567943C (en) | 2007-04-27 | 2007-04-27 | A kind of section statining method of skeletal muscle band |
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| CN 200710051996 CN100567943C (en) | 2007-04-27 | 2007-04-27 | A kind of section statining method of skeletal muscle band |
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| CN101769836B (en) * | 2010-01-25 | 2011-11-09 | 河南科技大学 | Pine needle section observation method |
| CN103415762A (en) * | 2011-01-10 | 2013-11-27 | 文塔纳医疗系统公司 | Hematoxylin staining method |
| JP5924624B2 (en) * | 2011-04-28 | 2016-05-25 | 国立研究開発法人理化学研究所 | Method for making biological material transparent and use thereof |
| WO2012161143A1 (en) | 2011-05-20 | 2012-11-29 | 独立行政法人理化学研究所 | Clarifying reagent for biological materials and use thereof |
| CN103257055A (en) * | 2013-04-23 | 2013-08-21 | 上海海洋大学 | Preparation method of hyriopsis cumingii pallium tissue slice |
| WO2015022883A1 (en) | 2013-08-14 | 2015-02-19 | 独立行政法人理化学研究所 | Composition for preparing biomaterial with excellent light-transmitting property, and use thereof |
| CN103512885A (en) * | 2013-10-11 | 2014-01-15 | 江苏省家禽科学研究所 | Method for distinguishing muscle fiber type of duck skeletal muscle |
| CN104764645A (en) * | 2015-04-27 | 2015-07-08 | 西华师范大学 | Amphibian phalanx paraffin section manufacturing method |
| CN106370501B (en) * | 2015-07-23 | 2019-11-19 | 纪常生 | Pathological wax block closing machine |
| CN112198036A (en) * | 2020-09-16 | 2021-01-08 | 佛山科学技术学院 | Improved immunohistochemical staining method for paraffin section of pig brain tissue |
| CN113588375B (en) * | 2021-08-20 | 2023-09-29 | 中科光华(西安)智能生物科技有限公司 | High-stability bone and bone tumor tissue microarray chip and preparation method thereof |
| CN114689407B (en) * | 2022-04-14 | 2023-05-19 | 四川大学华西医院 | Method for manufacturing animal micro tissue paraffin section |
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Non-Patent Citations (2)
| Title |
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| 动物组织H.E.染色石蜡切片技术的改进. 任成林等.河北北方学院学报（自然科学版），第23卷第1期. 2007 |
| 动物组织H.E.染色石蜡切片技术的改进. 任成林等.河北北方学院学报（自然科学版），第23卷第1期. 2007 * |
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