CN106771256A - Skeletal muscle tissue frozen section myosin ATPase stain kit - Google Patents
Skeletal muscle tissue frozen section myosin ATPase stain kit Download PDFInfo
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- CN106771256A CN106771256A CN201710049773.2A CN201710049773A CN106771256A CN 106771256 A CN106771256 A CN 106771256A CN 201710049773 A CN201710049773 A CN 201710049773A CN 106771256 A CN106771256 A CN 106771256A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
Abstract
The invention provides skeletal muscle tissue frozen section myosin adenosinetriphosphataes（ATPase）Staining kit, contains CaCl2 Cushioning liquid, acetate buffer, substrate Incubating Solution, 2wt.% cobalt chloride solutions, 1wt.% ammonium sulfide solutions.The present invention establishes a set of skeletal muscle tissue frozen section myosin ATPase stain kit, so easy to use, not only increases operating efficiency, shortens going out the report cycle for case, and also saves reagent, and cost is greatly lowered.And stablized and satisfied result.
Description
Technical field
The invention belongs to biological technical field, and in particular to skeletal muscle tissue frozen section myosin adenosinetriphosphataes
（ATPase）Staining kit.
Background technology
Myosin adenosinetriphosphataes（myosin ATPase）Be positioned at skeletal muscle, skeletal muscle fibre be divided into I types flesh and
II type fleshes, II type muscle fibres are divided into IIA, IIB, and under normal circumstances, I types, IIA, IIB type are distributed in mosaic.ATPase is dyeed
Such as there is situations below and consider pathological change：Muscle fiber types groupization, certain fiber type is dominant, and certain fiber type missing equal distribution is mixed
Disorderly；Certain fiber type atrophy or hypertrophy, muscle fibre are rounded or the size and shape such as streak changes；There is undifferentiated IIC
Type muscle fibre.There is IIC type muscle fibres in infant's skeletal muscle before normal 4-5 Sui, be undifferentiated muscle fibre, such as appear in into
In the biopsy skeletal muscle of people, point out have pathological change.Therefore, ATPase dyeing is differentiate muscle fiber types and distribution most preferable
Reaction enzymes, to neurotic atrophy diagnosis have important value.Conventional calcium activation method, is widely used at present.But in reality
Be there are problems that in the application of border many：Because dye liquor is reused, Color is poor, it is desirable to reagent matching while using, and accurate adjustment
The acid-base value of solution；But the need for dip method and detection pH value, reagent preparation amount should not be so both time-consuming very little, wastes again
Reagent.Therefore, the present invention by the practice more than 3 years with grope, improved on the basis of former method, establish a set of bone
Bone muscular tissue frozen section myosin ATPase stain kit, it is so easy to use, not only increase work effect
Rate, shortens going out the report cycle for case, and also saves reagent, and cost is greatly lowered.And stablized and be satisfied with
As a result.
The content of the invention
It is an object of the invention to provide skeletal muscle tissue frozen section myosin ATPase stain kit,
It is easy to use, operating efficiency is not only increased, going out the report cycle for case is shortened, and reagent is also saved, significantly drop
Low cost.And stablized and satisfied result.
To achieve the above object, the present invention is adopted the following technical scheme that：
Skeletal muscle tissue frozen section myosin ATPase stain（ATPase）Kit, the kit include with
Lower composition：
（1）No. 1 liquid, CaCl2 Cushioning liquid：0.1mol/L glycine solutions, 0.1mol/LNaCl solution, 1 mol/L
CaCl2, 0.1 mol/L NaOH, with 0.1mol/L NaOH regulation pH value to 9.5 ± 0.01；
(2) No. 2 liquid, acetate buffer：0.2 mol/L sodium acetates, 0.2 mol/L acetic acid is adjusted with 0.1mol/L HCl
PH value is to 4.6 ± 0.01；
(3) No. 3 liquid, substrate Incubating Solution：Take the CaCl in No. 1 liquid2Cushioning liquid 10ml, ATP- disodium salt 25mg, uses
0.1mol/L NaOH adjust pH to 9.4 ± 0.01；
（4）No. 4 liquid, 2wt.% cobalt chloride solutions；
（5）No. 5 liquid, ammonium sulfide solution：Ammonium sulfide working solution：1wt% ammonium sulfide solutions, are using preceding Fresh.
1-3 liquid is frozen in -20 DEG C, -35 DEG C of refrigerators or -80 DEG C of refrigerators；4th, No. 5 liquid is cold is stored in -4 DEG C of refrigerators.
It is using the colouring method of the kit including as follows：
（1）Muscle biopsy tissue, OCT embeddings, the isopentane through Liquid nitrogen precooler is quick-frozen, with cryostat in -22 DEG C of incisions
Piece, thickness is 6 μm, is stored in -20 DEG C or -35 DEG C of refrigerators；
（2）Take every frozen section two panels and be divided into two groups of A, B, electricity consumption blowing solid carbon dioxide steam；
（3）Alkaline preincubation：The section of A groups is added dropwise CaCl2 Cushioning liquid 100 μ L, the min of room temperature 10；
（4）Acid preincubation：The section of B groups is added dropwise acetate buffer 100 μ L, the min of room temperature 10, with filter paper by B group section groups
The acetate buffer for knitting surrounding is sopped up, and CaCl is added dropwise2 Cushioning liquid 100 μ L, room temperature 30sec；
（5）Substrate is incubated：With filter paper by the CaCl around two groups of biopsy tissues of A, B2 Cushioning liquid is sopped up, and is added dropwise to substrate and is incubated
Liquid 100ul is educated, 37 DEG C, 60 min is incubated；
（6）The substrate Incubating Solution around biopsy tissues is inhaled with filter paper；
（7）2wt.% cobalt chloride solution 0.5mL are added dropwise, 5 min are placed at room temperature；
（8）Running water rinses 3 min, and distilled water embathes 2 min；
（9）By in section insertion 1wt.% ammonium sulfide solutions, 1 min is soaked at room temperature；
（10）Flowing water rinses 5min；
（11）Will section by 95% ethanol, 100% ethanol dehydration, dimethylbenzene is transparent, after canada balsam sealing, dyeing is completed.
Myosin ATP in muscle fibre（Atriphos）Enzyme hydrolysis ATP is adenosine diphosphate (ADP) and phosphoric acid, and releases energy
Amount, phosphoric acid and calcium binding, form colourless calcium phosphate at enzymatic activity, and calcium phosphate is processed through cobalt chloride, forms cobalt phosphate,
The cobalt sulfide for forming brown through ammonium sulfide treatment again is deposited in zymophore.A groups are cut into slices in pH9.5 CaCl2Cushioning liquid
In alkaline environment during preincubate, it is suppressed that the atpase activity in I type muscle fibres, it is set not develop the color, and IIA, IIB type muscle fibre
Deep, light brown is shown respectively；The section of B groups inhibits II types in the sour environment of pH4.6 acetate buffers during preincubate, then
Atpase activity in muscle fibre, II type muscle fibres do not develop the color, and I types muscle fibre display dark-brown.In alkaline preincubation and acidity
The IIC equal non-inactivations of type muscle fibre after preincubation treatment, show light brown.
The reaction of each enzyme has its optimum pH, myosin adenosinetriphosphataes（ATPase）Optimum pH
It is 9.4, during higher or lower than optimum pH, the reaction speed of enzyme declines, the reaction result all effected of enzyme.From dyeing theory
With in result it will be evident that the pH value of preincubation liquid is also one of key factor for influenceing ATP enzyme to react in the method, it is preceding to incubate
The acid-base value for educating liquid is different, and the result of enzyme reaction is different.Therefore, it is accurate with substrate Incubating Solution pH value to ensure preincubation liquid
Degree, is all Fresh Incubating Solution before use in conventional method, and adjust pH value, it is seen then that one of key of the experiment is exactly molten
The acid-base value of liquid.Therefore, author is attempted using the method for Cord blood, and the extensive use of Cord blood article is such as cultivated thin
Born of the same parents, bacterium, antibody freeze.Low temperature slows down molecular motion velocities, and material composition composition is in metastable state, solution
Acid-base property be also held essentially constant.According to cryopreservation principle, author attempts to set up ATP enzyme（ATPase）Tissue section strain
Kit, and dip method is changed to drip dye method.Originally the amount of the minimum substrate Incubating Solution needed for first use is 10 ml, by it
Into 200ul/ ampoules after packing, 50 sets of kits are fabricated to, and frozen in -80 DEG C of low temperature refrigerators, using preceding thawing.Each
Section consumption 100ul, each person-portion sample does acid each with basic dyeing a piece of, and consumption is 200ul, so can be used for 50 person-portions
Muscle biopsy sample.
The advantage of the invention is that：The key of the kit is low dose of packing and freezes that, if do not dispense freezing, repetition makes
It is poor with effect.Frozen again after low dose packing, use more convenient and effect good.Preincubation liquid and the substrate for preparing are incubated
Liquor is educated to freeze in -80 DEG C of refrigerators, storage life 1 year.The reagent for freezing experimental result and fresh reagent matched somebody with somebody in storage life
Experimental result is consistent.The foundation of the kit can save trouble every time using preceding Fresh dye liquor, regulation acid-base value etc., save
When it is easy, not only increase operating efficiency, and also save reagent, cost can be greatly lowered.Pathology can additionally be shortened
Interval between diagnosis, obtained medical treatment very important effect in time to patient.This kit is by author to the clinic of 300 many cases
The practical application of Muscle biopsy sample and checking, show the foundation of the kit, and method is feasible, reliable results, and can promote should
With.
In this dyeing procedure, original dip method is changed to drip dye method, it is important to（1）Substrate Incubating Solution will be from before dyeing
Refrigerator is taken out, and 2000 revs/min are carried out after thawing, and is centrifuged 10 minutes, and Aspirate supernatant is used, better.（2）Section is adding
Before substrate Incubating Solution or after the incubation of substrate Incubating Solution, can not be rinsed with water.（3）It is dry with originally washing after cobalt chloride dyeing
Only, otherwise, cut into slices whole nigrescences after adding ammonium sulfide；（4）Ammonium sulfide solution 3 min dilutions before colour developing, and add a cover.Vulcanization
The resting period of ammonium stoste is too long, can voluntarily decompose and fail, and when the smell without ammonia, it is necessary to change the liquid, otherwise influences
Coloration result.
Brief description of the drawings
Fig. 1 ATPase dyeing × 200；A groups are cut into slices（Alkaline preincubation group）I type muscle fibres do not develop the color, II type muscle fibres
Colour developing.Amphitypy flesh is in inserted arrangement.
Fig. 2：ATPase dyeing × 200；B groups are cut into slices（Acid preincubation group）I types muscle fibre develops the color, and II types muscle fibre is not
Colour developing.Amphitypy flesh is in inserted arrangement.
Fig. 3：ATPase dyeing × 200；A groups are cut into slices（Alkaline preincubation group）I type muscle fibres do not develop the color, and II types flesh is fine
Dimension colour developing.Amphitypy flesh is in inserted arrangement.Reagent（- 80 DEG C freeze 1 year）
Fig. 4：ATPase dyeing × 200；B groups are cut into slices（Acid preincubation group）I types muscle fibre develops the color, and II type muscle fibres do not show
Color.Amphitypy flesh is in inserted arrangement.Reagent（- 80 DEG C freeze 1 year）
Fig. 5：ATPase dyeing × 200；A groups are cut into slices（Alkaline preincubation group）I type muscle fibres do not develop the color, the colour developing of II types muscle fibre.
Atrophy fiber is II types, and hypertrophic fibers are I types, and muscle fibre is presented homotype muscle group.
Fig. 6：ATPase dyeing × 200；B groups are cut into slices（Acid preincubation group）I types muscle fibre develops the color, and II types muscle fibre is not
Colour developing.Atrophy fiber is II types, and hypertrophic fibers are I types, and muscle fibre is presented homotype muscle group.
Fig. 7：ATPase dyeing × 200；A groups are cut into slices（Alkaline preincubation group）I type muscle fibres do not develop the color, and II types flesh is fine
Dimension colour developing.Amphitypy flesh is in inserted arrangement.Reagent（Fresh）.
Fig. 8：ATPase dyeing × 200；B groups are cut into slices（Acid preincubation group）I types muscle fibre develops the color, II type muscle fibres
Do not develop the color.Amphitypy flesh is in inserted arrangement.Reagent（Fresh）.
Specific embodiment
Embodiment 1
Skeletal muscle tissue frozen section myosin adenosinetriphosphataes（ATPase）Staining kit, the kit include with
Lower composition：
（1）CaCl2 Cushioning liquid（No. 1 liquid）：0.1mol/L glycine solutions, 0.1mol/LNaCl solution, 1 mol/L
CaCl2, 0.1 mol/L NaOH, with 0.1mol/L NaOH regulation pH value to 9.5 ± 0.01；
(2) acetate buffer（No. 2 liquid）：0.2 mol/L sodium acetates, 0.2 mol/L acetic acid, use 0.1mol/L HCl
Adjust pH value to 4.6 ± 0.01；
(3) substrate Incubating Solution（No. 3 liquid）：Take the CaCl in No. 1 liquid2Cushioning liquid 10ml, ATP- disodium salt 25mg, uses
0.1mol/L NaOH adjust pH to 9.4 ± 0.01；
（4）2wt.% cobalt chloride solutions（No. 4 liquid）；
（5）Ammonium sulfide solution（No. 5 liquid）：Ammonium sulfide working solution：1wt% ammonium sulfide solutions, are using preceding Fresh.
1-3 liquid is frozen in -20 DEG C, -35 DEG C of refrigerators or -80 DEG C of refrigerators；4th, No. 5 liquid is cold is stored in -4 DEG C of refrigerators.
It is using the colouring method of the kit including as follows：
（1）Muscle biopsy tissue, OCT embeddings, the isopentane through Liquid nitrogen precooler is quick-frozen, with cryostat in -22 DEG C of incisions
Piece, thickness is 6 μm, is stored in -20 DEG C or -35 DEG C of refrigerators；
（2）Take every frozen section two panels and be divided into two groups of A, B, electricity consumption blowing solid carbon dioxide steam；
（3）Alkaline preincubation：The section of A groups is added dropwise CaCl2 Cushioning liquid 100 μ L, the min of room temperature 10；
（4）Acid preincubation：The section of B groups is added dropwise acetate buffer 100 μ L, the min of room temperature 10, with filter paper by B group section groups
The acetate buffer for knitting surrounding is sopped up, and CaCl is added dropwise2 Cushioning liquid 100 μ L, room temperature 30sec；
（5）Substrate is incubated：With filter paper by the CaCl around two groups of biopsy tissues of A, B2 Cushioning liquid is sopped up, and is added dropwise to substrate and is incubated
Liquid 100ul is educated, 37 DEG C, 60 min is incubated；
（6）The substrate Incubating Solution around biopsy tissues is sopped up with filter paper；
（7）2wt.% cobalt chloride solution 0.5mL are added dropwise, 5 min are placed at room temperature；
（8）Running water rinses 3 min, and distilled water embathes 2 min；
（9）By in section insertion 1wt.% ammonium sulfide solutions, 1 min is soaked at room temperature；
（10）Flowing water rinses 5min；
（11）Will section by 95% ethanol, 100% ethanol dehydration, dimethylbenzene is transparent, after canada balsam sealing, dyeing is completed.
Application Example：
Case 1：
Patient, man, 15 years old, is chief complaint with gradually progressive weakness of limbs and is admitted to hospital, and is had a medical check-up：Four limbs muscular strength declines, and muscular atrophy is bright
It is aobvious.Clinical diagnosis is Myopathic lesion.Carry out tissue biopsy, biopsy site：LD musculature.
As a result：Basis of microscopic observation：ATPase dyeing amphitypy muscle fibres are in inserted arrangement, and muscle fibre is shown in polygonal
It is dispersed in the slight atrophy of muscle fibre（Such as Fig. 1-2）.
Pathological diagnosis：Muscular tissue has no that specific pathologies change.
Case 2：
Patient, man, 33 years old.It is chief complaint within 4 months with gradual limb adynamia and is admitted to hospital.Have a medical check-up：Left lower extremity left upper extremity muscular atrophy.Face
Bed diagnosis：Myasthenia unknown origin.Carry out tissue biopsy, biopsy site：The left bicipital muscle of arm.
As a result：Basis of microscopic observation：ATPase dyeing is shown in that muscle fibre differs in size, and loose and bar shaped atrophy muscle fibre is alternate
Arrangement, amphitypy muscle fibre is in inserted arrangement, has no homotype muscle group, and amphitypy muscle fibre has atrophy.（See Fig. 3-4）
Pathological diagnosis：Neurogenic muscular atrophy.
Case 3：
Patient, female 2 months 1 years old, is chief complaint and is admitted to hospital for powerless 7 months with double lower limb progressive.Have a medical check-up：Four limbs myasthenia is obvious.
Carry out tissue biopsy, biopsy site：Right quadriceps muscle of thigh.
As a result：Basis of microscopic observation：ATPase is dyeed：See the muscle fibre for diffusing circular atrophy, be dispersed in obvious hypertrophy
Muscle fibre is mingled with wherein, and based on II types, hypertrophic fibers are I types to atrophy fiber, and muscle fibre is presented homotype muscle group.（See Fig. 5-
6）
Pathological diagnosis：Spinal muscular atrophy.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to covering scope of the invention.
Claims (2)
1. skeletal muscle tissue frozen section myosin ATPase stain kit, it is characterised in that：The kit
Comprising following component：
（1）No. 1 liquid, CaCl2 Cushioning liquid：0.1 mol/L glycine solutions, 0.1 mol/LNaCl solution, 1 mol/L
CaCl2, 0.1 mol/L NaOH, with 0.1mol/L NaOH regulation pH value to 9.5 ± 0.01；
(2) No. 2 liquid, acetate buffer：0.2 mol/L sodium acetates, 0.2 mol/L acetic acid is adjusted with 0.1mol/L HCl
PH value is to 4.6 ± 0.01；
(3) No. 3 liquid, substrate Incubating Solution：Take the CaCl in No. 1 liquid2Cushioning liquid 10ml, ATP- disodium salt 25mg, uses 0.1mol/
L NaOH adjust pH to 9.4 ± 0.01；
（4）No. 4 liquid, 2wt.% cobalt chloride solutions；
（5）No. 5 liquid, ammonium sulfide solution：1wt% ammonium sulfide solutions, are using preceding Fresh.
2. such as the colouring method of claim 1 kit as described in, it is characterised in that：Methods described includes as follows：
（1）Muscle biopsy tissue, OCT embeddings, the isopentane through Liquid nitrogen precooler is quick-frozen, with cryostat in -22 DEG C of incisions
Piece, thickness is 6 μm, is stored in -20 DEG C or -35 DEG C of refrigerators；
（2）Take every frozen section two panels and be divided into two groups of A, B, electricity consumption blowing solid carbon dioxide steam；
（3）Alkaline preincubation：The section of A groups is added dropwise CaCl2 Cushioning liquid 100 μ L, the min of room temperature 10；
（4）Acid preincubation：The section of B groups is added dropwise acetate buffer 100 μ L, the min of room temperature 10, with filter paper by B group section groups
The acetate buffer for knitting surrounding is sopped up, and CaCl is added dropwise2 Cushioning liquid 100 μ L, room temperature 30sec；
（5）Substrate is incubated：With filter paper by the CaCl around two groups of biopsy tissues of A, B2 Cushioning liquid is sopped up, and is added dropwise to substrate incubation
Liquid 100ul, is incubated 60 min by 37 DEG C；
（6）The substrate Incubating Solution around biopsy tissues is sopped up with filter paper；
（7）2wt.% cobalt chloride solution 0.5mL are added dropwise, 5 min are placed at room temperature；
（8）Running water rinses 3 min, and distilled water embathes 2 min；
（9）By in section insertion 1wt.% ammonium sulfide solutions, 1 min is soaked at room temperature；
（10）Flowing water rinses 5min；
（11）Will section by 95% ethanol, 100% ethanol dehydration, dimethylbenzene is transparent, after canada balsam sealing, dyeing is completed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107831112A (en) * | 2017-09-22 | 2018-03-23 | 浙江海洋大学 | A kind of Histological method for distinguishing three kinds of muscle cells of carp |
| CN113008647A (en) * | 2021-02-25 | 2021-06-22 | 武汉魅杰生物科技有限公司 | ATP enzyme histochemical staining method |
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| EP1860186A2 (en) * | 2003-03-10 | 2007-11-28 | Japan Science and Technology Corporation | Method for distinguishing mesenchymal stem cells using different markers |
| CN102288601A (en) * | 2011-05-16 | 2011-12-21 | 中国药科大学 | Method for microdetermination of ATPase activity of myoglobulin and application of Ruscogenin |
| 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 |
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2017
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| EP1860186A2 (en) * | 2003-03-10 | 2007-11-28 | Japan Science and Technology Corporation | Method for distinguishing mesenchymal stem cells using different markers |
| CN102288601A (en) * | 2011-05-16 | 2011-12-21 | 中国药科大学 | Method for microdetermination of ATPase activity of myoglobulin and application of Ruscogenin |
| 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 |
Non-Patent Citations (3)
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| 张巧全等: "肌肉病理检查中ATP酶(pH9.4)和ATP酶(pH4.6)染色的合并孵育", 《临床神经病学杂志》 * |
| 张萌等: "ATP酶组织化学滴染法染色技术", 《解剖学研究》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107831112A (en) * | 2017-09-22 | 2018-03-23 | 浙江海洋大学 | A kind of Histological method for distinguishing three kinds of muscle cells of carp |
| CN113008647A (en) * | 2021-02-25 | 2021-06-22 | 武汉魅杰生物科技有限公司 | ATP enzyme histochemical staining method |
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| CN106771256B (en) | 2019-02-01 |
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