CN106771256B - Skeletal muscle tissue frozen section myosin ATPase stain method - Google Patents

Skeletal muscle tissue frozen section myosin ATPase stain method Download PDF

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CN106771256B
CN106771256B CN201710049773.2A CN201710049773A CN106771256B CN 106771256 B CN106771256 B CN 106771256B CN 201710049773 A CN201710049773 A CN 201710049773A CN 106771256 B CN106771256 B CN 106771256B
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cacl
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CN106771256A (en
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高美钦
刘文文
陈裕庆
晋雯
张文敏
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Fujian Medical University
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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
    • 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
    • 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
    • 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 present invention provides skeletal muscle tissue frozen section myosin adenosinetriphosphataes (ATPase) staining kits, contain CaCl2Buffer solution, acetate buffer, substrate Incubating Solution, 2wt.% cobalt chloride solution, 1wt.% ammonium sulfide solution.The present invention establishes a set of skeletal muscle tissue frozen section myosin ATPase stain kit, easy to use in this way, not only increases working efficiency, and shorten case goes out reporting cycle, and also saves reagent, and cost is greatly lowered.And the result stablized and be satisfied with.

Description

Skeletal muscle tissue frozen section myosin ATPase stain method
Technical field
The invention belongs to field of biotechnology, and in particular to skeletal muscle tissue frozen section myosin adenosinetriphosphataes (ATPase) staining kit.
Background technique
Myosin adenosinetriphosphataes (myosin ATPase) are positioned at skeletal muscle, skeletal muscle fibre be divided into I type flesh and II type flesh, II type muscle fibre are divided into IIA, IIB, and under normal circumstances, I type, IIA, IIB type are distributed in mosaic.ATPase dyeing Such as there is following situations and consider pathological change: muscle fiber types groupization, certain fiber type is dominant, and it is mixed that certain fiber type lacks equal distribution Disorderly；Certain fiber type atrophy or hypertrophy, muscle fibre are rounded or the size and shapes such as streak change；There is undifferentiated IIC Type muscle fibre.Normally there are IIC type muscle fibres in infant's skeletal muscle before 4-5 years old, are undifferentiated muscle fibre, such as appear in into In the biopsy skeletal muscle of people, prompt have pathological change.Therefore, ATPase dyeing is differentiate muscle fiber types and distribution most ideal Reaction enzymes, to neurotic atrophy diagnosis have important value.Common calcium activation method at present, is widely used.But in reality There is many problems in the application of border: because dye liquor is reused, dyeing effect is poor, it is desirable that reagent matching while using, and accurately adjust The pH value of solution；However dip method and the needs for detecting pH value, reagent preparation amount should not be very little, and it is not only time-consuming in this way, but also waste Reagent.Therefore, the present invention by more than 3 years practice with grope, improved on the basis of original method, establish a set of bone Bone muscular tissue frozen section myosin ATPase stain kit, it is easy to use in this way, not only increase work effect Rate, shorten case goes out reporting cycle, and also saves reagent, and cost is greatly lowered.And stablized and satisfied As a result.
Summary of the invention
The purpose of the present invention is to provide skeletal muscle tissue frozen section myosin ATPase stain kit, It is easy to use, working efficiency is not only increased, shorten case goes out reporting cycle, and also saves reagent, significantly drops Low cost.And the result stablized and be satisfied with.
To achieve the above object, the present invention adopts the following technical scheme:
Skeletal muscle tissue frozen section myosin ATPase stain (ATPase) kit, the kit packet Containing following component:
(1) No. 1 liquid, CaCl2 Buffer solution: 0.1mol/L glycine solution, 0.1mol/LNaCl solution, 1 mol/L CaCl2, 0.1 mol/L NaOH, with 0.1mol/L NaOH adjusting pH value to 9.5 ± 0.01；
(2) No. 2 liquid, acetate buffer: 0.2 mol/L sodium acetate, 0.2 mol/L acetic acid use 0.1mol/L HCl tune pH value is to 4.6 ± 0.01；
(3) No. 3 liquid, substrate Incubating Solution: take the CaCl in No. 1 liquid2Buffer solution 10ml, ATP- disodium salt 25mg is used 0.1mol/L NaOH tune pH to 9.4 ± 0.01；
(4) No. 4 liquid, 2wt.% cobalt chloride solution；
(5) No. 5 liquid, ammonium sulfide solution: ammonium sulfide working solution: 1wt% ammonium sulfide solution, the Fresh before use.
1-3 liquid freezes in -20 DEG C, -35 DEG C of refrigerators or -80 DEG C of refrigerators；4, No. 5 liquid coolings are stored in -4 DEG C of refrigerators.
Using the colouring method of the kit, include the following:
(1) Muscle biopsy tissue, OCT embedding, the isopentane through Liquid nitrogen precooler is quick-frozen, with cryostat at -22 DEG C Lower slice is stored in -20 DEG C or -35 DEG C of refrigerators with a thickness of 6 μm；
(2) every frozen section two panels is taken to be divided into two groups of A, B, electricity consumption blowing solid carbon dioxide steam；
(3) alkaline preincubation: A group is sliced, CaCl is added dropwise2 Buffer solution 100 μ L, 10 min of room temperature；
(4) acid preincubation: B group is sliced, 100 μ L of acetate buffer, 10 min of room temperature is added dropwise, cut B group with filter paper Acetate buffer around piece tissue sops up, and CaCl is added dropwise2 Buffer solution 100 μ L, room temperature 30sec；
(5) substrate is incubated for: with filter paper by the CaCl around two groups of biopsy tissues of A, B2 Buffer solution sops up, and is added dropwise to substrate Incubating Solution 100ul, is incubated for 60 min by 37 DEG C；
(6) the substrate Incubating Solution around biopsy tissues is inhaled with filter paper；
(7) 2wt.% cobalt chloride solution 0.5mL is added dropwise, places 5 min at room temperature；
(8) tap water rinses 3 min, and distilled water embathes 2 min；
(9) by slice insertion 1wt.% ammonium sulfide solution, 1 min is impregnated at room temperature；
(10) flowing water rinses 5min；
(11) will slice by 95% ethyl alcohol, 100% ethanol dehydration, dimethylbenzene is transparent, after canada balsam sealing, dyed At.
Myosin ATP(atriphos in muscle fibre) 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 handled through cobalt chloride, form cobalt phosphate, The cobalt sulfide for forming brown through ammonium sulfide processing again is deposited in zymophore.A group is sliced in pH9.5 CaCl2Buffer solution In alkaline environment when preincubate, it is suppressed that the atpase activity in I type muscle fibre makes it not develop the color, and IIA, IIB type muscle fibre Deep, light brown is shown respectively；B group slice when preincubate, then inhibits II type in the acidic environment of pH4.6 acetate buffer Atpase activity in muscle fibre, II type muscle fibre do not develop the color, and I type muscle fibre shows dark-brown.In alkaline preincubation and acidity The equal non-inactivation of IIC type muscle fibre after preincubation processing, shows light brown.
The reaction of each enzyme has its optimum pH, the optimum pH of myosin adenosinetriphosphataes (ATPase) It is 9.4, when being 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 to influence one of the key factor reacted of ATP enzyme in this method, it is preceding to incubate The pH value for educating liquid is different, and the result of enzyme reaction is different.Therefore, to guarantee the accurate of preincubation liquid and substrate Incubating Solution pH value Degree is all Fresh Incubating Solution before use in conventional method, and adjusts pH value, it is seen then that one of key of the experiment is exactly molten The pH value of liquid.Therefore, author attempts the method for utilizing cryo-conservation, and cryo-conservation article has been widely applied, and such as cultivates thin Born of the same parents, bacterium, antibody freeze.Low temperature slows down molecular motion velocities, and material composition ingredient is in metastable state, solution Acid-base property be also held essentially constant.According to cryopreservation principle, author attempts to establish ATP enzyme (ATPase) tissue section strain Kit, and dip method is changed to drop dye method.Originally the amount of minimum substrate Incubating Solution needed for first use is 10 ml, by it At 200ul/ ampoule after packing, 50 sets of kits are fabricated to, and are frozen in -80 DEG C of low temperature refrigerators, are thawed before use.Each It is sliced dosage 100ul, each person-portion sample does acid each a piece of with basic dyeing, and dosage 200ul can be used for 50 person-portions in this way Muscle biopsy sample.
The present invention has the advantages that the key of the kit is low dose of packing and freezes, frozen if not dispensing, repetition makes It is poor with effect.It is frozen again after low dose packing, it is good to use more convenient and effect.Prepared preincubation liquid is incubated with substrate It educates liquor to freeze in -80 DEG C of refrigerators, storage life 1 year.The reagent frozen experimental result and fresh reagent matched in storage life Experimental result is consistent.The foundation of the kit can save every time using the trouble of preceding Fresh dye liquor, adjusting pH value etc., save When it is easy, not only increase working efficiency, and also save reagent, cost can be greatly lowered.Pathology can additionally be shortened Interval between diagnosis, obtained medical treatment in time to patient very important effect.This kit is by author to the clinic of 300 many cases The practical application and verifying of Muscle biopsy sample, show the foundation of the kit, and method is feasible, as a result reliably, can promote and answer With.
In this dyeing procedure, original dip method is changed to drop dye method, it is important to which (1) substrate Incubating Solution will be from before dyeing Refrigerator takes out, and 2000 revs/min are carried out after thawing, and is centrifuged 10 minutes, and Aspirate supernatant uses, better effect.(2) slice is adding Before substrate Incubating Solution or after the incubation of substrate Incubating Solution, cannot it be rinsed with water.(3) dry with originally washing after cobalt chloride dyeing Only, otherwise, it is sliced after ammonium sulfide is added by whole nigrescences；(4) ammonium sulfide solution 3 min before colour developing dilute, and cover.Vulcanization The resting period of ammonium stoste is too long, can voluntarily decompose and fail, when the not smell of ammonia, need replacing the liquid, otherwise influence Coloration result.
Detailed description of the invention
Fig. 1 ATPase dyeing × 200；A group slice (alkaline preincubation group) I type muscle fibre does not develop the color, II type muscle fibre Colour developing.Amphitypy flesh is in inserted arrangement.
Fig. 2: ATPase dyeing × 200；B group is sliced the colour developing of (acid preincubation group) I type muscle fibre, and II type muscle fibre is not Colour developing.Amphitypy flesh is in inserted arrangement.
Fig. 3: ATPase dyeing × 200；A group slice (alkaline preincubation group) I type muscle fibre does not develop the color, and II type 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 group is sliced the colour developing of (acid preincubation group) I type muscle fibre, II type muscle fibre It does not develop the color.Amphitypy flesh is in inserted arrangement.Reagent (- 80 DEG C freeze 1 year)
Fig. 5: ATPase dyeing × 200；A group slice (alkaline preincubation group) I type muscle fibre does not develop the color, II type muscle fibre Colour developing.Atrophy fiber is II type, and hypertrophic fibers are I type, and homotype muscle group is presented in muscle fibre.
Fig. 6: ATPase dyeing × 200；B group is sliced the colour developing of (acid preincubation group) I type muscle fibre, and II type muscle fibre is not Colour developing.Atrophy fiber is II type, and hypertrophic fibers are I type, and homotype muscle group is presented in muscle fibre.
Fig. 7: ATPase dyeing × 200；A group slice (alkaline preincubation group) I type muscle fibre does not develop the color, and II type flesh is fine Dimension colour developing.Amphitypy flesh is in inserted arrangement.Reagent (Fresh).
Fig. 8: ATPase dyeing × 200；B group is sliced the colour developing of (acid preincubation group) I type muscle fibre, II type muscle fibre It does 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 packet Containing following component:
(1) CaCl2 Buffer solution (No. 1 liquid): 0.1mol/L glycine solution, 0.1mol/LNaCl solution, 1 mol/L CaCl2, 0.1 mol/L NaOH, with 0.1mol/L NaOH adjusting pH value to 9.5 ± 0.01；
(2) acetate buffer (No. 2 liquid): 0.2 mol/L sodium acetate, 0.2 mol/L acetic acid use 0.1mol/L HCl tune pH value is to 4.6 ± 0.01；
(3) substrate Incubating Solution (No. 3 liquid): the CaCl in No. 1 liquid is taken2Buffer solution 10ml, ATP- disodium salt 25mg is used 0.1mol/L NaOH tune pH to 9.4 ± 0.01；
(4) 2wt.% cobalt chloride solution (No. 4 liquid)；
(5) ammonium sulfide solution (No. 5 liquid): ammonium sulfide working solution: 1wt% ammonium sulfide solution, the Fresh before use.
1-3 liquid freezes in -20 DEG C, -35 DEG C of refrigerators or -80 DEG C of refrigerators；4, No. 5 liquid coolings are stored in -4 DEG C of refrigerators.
Using the colouring method of the kit, include the following:
(1) Muscle biopsy tissue, OCT embedding, the isopentane through Liquid nitrogen precooler is quick-frozen, with cryostat at -22 DEG C Lower slice is stored in -20 DEG C or -35 DEG C of refrigerators with a thickness of 6 μm；
(2) every frozen section two panels is taken to be divided into two groups of A, B, electricity consumption blowing solid carbon dioxide steam；
(3) alkaline preincubation: A group is sliced, CaCl is added dropwise2 Buffer solution 100 μ L, 10 min of room temperature；
(4) acid preincubation: B group is sliced, 100 μ L of acetate buffer, 10 min of room temperature is added dropwise, cut B group with filter paper Acetate buffer around piece tissue sops up, and CaCl is added dropwise2 Buffer solution 100 μ L, room temperature 30sec；
(5) substrate is incubated for: with filter paper by the CaCl around two groups of biopsy tissues of A, B2 Buffer solution sops up, and is added dropwise to substrate Incubating Solution 100ul, is incubated for 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 is added dropwise, places 5 min at room temperature；
(8) tap water rinses 3 min, and distilled water embathes 2 min；
(9) by slice insertion 1wt.% ammonium sulfide solution, 1 min is impregnated at room temperature；
(10) flowing water rinses 5min；
(11) will slice by 95% ethyl alcohol, 100% ethanol dehydration, dimethylbenzene is transparent, after canada balsam sealing, dyed At.
Application Example:
Case 1:
Patient, male, 15 years old, is chief complaint with gradually progressive weakness of limbs and is admitted to hospital, physical examination: the decline of four limbs muscular strength, muscle wither Contracting is obvious.Clinical diagnosis is Myopathic lesion.Tissue biopsy is carried out, biopsy site: left deltoid muscle musculature.
As a result: microscopically observation: it is in inserted arrangement that ATPase, which dyes amphitypy muscle fibre, 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, male, 33 years old.It is chief complaint within 4 months and is admitted to hospital with gradual limb adynamia.Physical examination: left lower extremity left upper extremity muscle withers Contracting.Clinical diagnosis: myasthenia unknown origin.Tissue biopsy is carried out, biopsy site: the left bicipital muscle of arm.
As a result: microscopically 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 are in inserted arrangement, have no that homotype muscle group, amphitypy muscle fibre have atrophy.(see Fig. 3-4)
Pathological diagnosis: neurogenic muscular atrophy.
Case 3:
Patient, female 2 months 1 years old, are powerlessly chief complaint with double lower limb progressive and are admitted to hospital for 7 months.Physical examination: four limbs myasthenia is bright It is aobvious.
Tissue biopsy is carried out, biopsy site: right quadriceps muscle of thigh.
As a result: microscopically observation: ATPase dyeing: being shown in the muscle fibre for diffusing round atrophy, is dispersed in obvious hypertrophy Muscle fibre is mingled with wherein, and for atrophy fiber based on II type, hypertrophic fibers are I type, and homotype muscle group is presented in muscle fibre.(see Fig. 5- 6)
Pathological diagnosis: spinal muscular atrophy.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

Claims (1)

1. the colouring method of skeletal muscle tissue frozen section myosin ATPase stain kit, it is characterised in that: Colouring method includes the following:
(1) Muscle biopsy tissue, OCT embedding, the isopentane through Liquid nitrogen precooler is quick-frozen, with cryostat in -22 DEG C of incisions Piece is stored in -20 DEG C or -35 DEG C of refrigerators with a thickness of 6 μm；
(2) every frozen section two panels is taken to be divided into two groups of A, B, electricity consumption blowing solid carbon dioxide steam；
(3) alkaline preincubation: A group is sliced, CaCl is added dropwise2 Buffer solution 100 μ L, 10 min of room temperature；
(4) acid preincubation: by B group slice dropwise addition 100 μ L of acetate buffer, 10 min of room temperature, with filter paper by B group slice group The acetate buffer for knitting surrounding sops up, and CaCl is added dropwise2 Buffer solution 100 μ L, room temperature 30sec；
(5) substrate is incubated for: with filter paper by the CaCl around two groups of biopsy tissues of A, B2 Buffer solution sops up, and is added dropwise to substrate incubation Liquid 100ul, is incubated for 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 is added dropwise, places 5 min at room temperature；
(8) tap water rinses 3 min, and distilled water embathes 2 min；
(9) by slice insertion 1wt.% ammonium sulfide solution, 1 min is impregnated at room temperature；
(10) flowing water rinses 5min；
(11) will slice by 95% ethyl alcohol, 100% ethanol dehydration, dimethylbenzene is transparent, after canada balsam sealing, dyeing is completed；
The kit includes following component:
(1) No. 1 liquid, CaCl2 Buffer solution: 0.1 mol/L glycine solution, 0.1 mol/LNaCl solution, 1 mol/L CaCl2, 0.1 mol/L NaOH, with 0.1mol/L NaOH adjusting pH value to 9.5 ± 0.01；
(2) No. 2 liquid, acetate buffer: 0.2 mol/L sodium acetate, 0.2 mol/L acetic acid, with 0.1mol/L HCl tune PH value is to 4.6 ± 0.01；
(3) No. 3 liquid, substrate Incubating Solution: take the CaCl in No. 1 liquid2Buffer solution 10ml, ATP- disodium salt 25mg, uses 0.1mol/ L NaOH tune pH to 9.4 ± 0.01；
(4) No. 4 liquid, 2wt.% cobalt chloride solution；
(5) No. 5 liquid, ammonium sulfide solution: 1wt% ammonium sulfide solution, the Fresh before use.
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CN107831112B (en) * 2017-09-22 2020-11-10 浙江海洋大学 Histology method for distinguishing three muscle cells of carp
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