CN104663646A - Embryonic tissue fixative and its preparation method and use - Google Patents
Embryonic tissue fixative and its preparation method and use Download PDFInfo
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- CN104663646A CN104663646A CN201310613171.7A CN201310613171A CN104663646A CN 104663646 A CN104663646 A CN 104663646A CN 201310613171 A CN201310613171 A CN 201310613171A CN 104663646 A CN104663646 A CN 104663646A
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- embryonic tissue
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Abstract
The invention provides an embryonic tissue fixative. The embryonic tissue fixative comprises, by mass, 0.67-0.79% of 2,4,6-trinitrophenol, 11-15.4% of formaldehyde, 0.38-0.58% of chromic acid, 4.2-6.8% of acetic acid and the balance water. The embryonic tissue fixative has fast penetration effects on a membrane structure and realizes fast penetration sizing, and protection and organization form preservation of embryonic tissue nucleic acid and protein. The embryonic tissue fixative can keep appropriate hardness of the embryonic tissue. Through the embryonic tissue fixative, complete continuous slices can be obtained easily and are convenient for later molecular pathological experiments. The invention also provides a preparation method and use of the embryonic tissue fixative.
Description
Technical field
The present invention relates to biological medicine Material Field, be specifically related to a kind of embryonic tissue fixative and its preparation method and application.
Background technology
The making of Pathologic specimen and histologic section need first to be fixed.Fixed effect is bad, and HE dyeing, SABC, in situ hybridization are just difficult to carry out, and therefore, tissue is fixedly indispensable important step in Senile Mouse.Existing histologic fixatives has aldehyde fixative (formalin, paraformaldehyde, glutaraldehyde etc.), precipitation dehydration class fixative (methyl alcohol, ethanol, acetone etc.), acids fixative (chromic acid, tannic acid, glacial acetic acid etc.), Bouin fixative, ZenkerShi fixative, CarrnoyShi fixative.
Embryonic tissue has its specific Structure and form, and such as embryo inside contains comparatively multimembrane tissue, and internal membrane tissue, containing more lipopolysaccharide composition, when embryonic tissue fixed by existing fixative, there will be various inferior position and shortcoming.Such as: when the chromic acid in acids fixative, tannic acid are used alone, infiltration is relatively slower, the fixing effect of fast shaping can not be played for Vitro Embryo tissue; Precipitation dehydration class fixative has the effect making Material shrinkage, and easily cause metaplasia, during section, hardness is too high, easily the shortcomings such as cavitation phenomena occurs, easily causes section to occur false positive results, therefore be unsuitable for the fixative needing to do embryonic tissue.
Therefore, be necessary to provide a kind of embryonic tissue fixative and preparation method thereof.
Summary of the invention
For solving the problem, the invention provides a kind of embryonic tissue fixative, this embryonic tissue fixative adopts compound prescription, containing 2,4,6-trinitrophenol, formaldehyde, chromic acid and acetic acid, this embryonic tissue fixative has rapid osmotic effect to membranous structure, can rapid osmotic sizing, the preservation on embryonic tissue nucleic acid, the protection of albumen and tissue morphology can be taken into account; The hardness that this embryonic tissue fixative also can make embryonic tissue keep suitable, easily obtains the section of complete continuous print, avoids cavitation phenomena, thus effectively can observe the tissue morphology in embryo development procedure, reduce the probability occurring false positive results.Present invention also offers a kind of preparation method and application of embryonic tissue fixative.
First aspect, the invention provides a kind of embryonic tissue fixative, and this embryonic tissue fixative contains 2,4,6-trinitrophenol, formaldehyde, chromic acid and acetic acid;
The percentage by weight of each component of described embryonic tissue fixative is:
2,4,6-trinitrophenol 0.67% ~ 0.79%;
Formaldehyde 11% ~ 15.4%;
Chromic acid 0.38% ~ 0.58%;
Acetic acid 4.2% ~ 6.8%;
Surplus is water.
Preferably, the percentage by weight of each component of described embryonic tissue fixative is:
2,4,6-trinitrophenol 0.73%;
Formaldehyde 13.2%;
Chromic acid 0.48%;
Acetic acid 5.1%;
Surplus is water.
Preferably, the percent by volume of each component of described fixative is:
Saturated 2,4,6-trinitrobenzen phenol solutions (mass fraction is 1.2%) 60%;
Formaldehyde (volume fraction 40%) 30%;
Saturated chromic acid solution (mass fraction is 12%) 5%;
Acetic acid (100%) 5%;
Particularly, in practical application, the formula of embryonic tissue fixative provided by the invention is not limited to acetic acid of the present invention, as required, and substituting of the acetic acid that at least one in other weak organic acids or other weak organic acids and acetic acid can adopt as the present invention.
Embryo contains comparatively multimembrane tissue, its internal membrane tissue is containing more lipopolysaccharide composition, fixative provided by the invention adopts and meets formula, embryo's internal membrane tissue can be permeated quickly and evenly, and reduce the appearance of cavitation phenomena, thus well preserve sample reset condition, and obtain complete pathologic tissue section.
In addition, embryonic tissue fixative provided by the invention reduces to the covering of proteantigen epi-position or destruction in fixation procedure, has higher protective efficacy for albumen, does not affect section and is used for doing SABC Pathological experiment.
The section that this fixative adopts fixative provided by the invention to prepare, after certain step process, can be used in RT-PCR, in situ hybridization, SABC, immunofluorescence equimolecular pathological experiment.
Chromic acid in embryonic tissue fixative provided by the invention is the good fixative of protein, also can help to preserve carbohydrate, but its penetration is lower, and acetic acid permeability in embryonic tissue fixative is strong, improves the fixing osmotic effect of fixative to embryonic tissue.
Picric acid (i.e. 2,4,6-trinitrophenols) can make the precipitation such as protein, nucleic acid, and prevents overvulcanization, can improve the colorability organized in follow-up dyeing course, but picric acid infiltration is in the tissue slow, and can make to organize strong contraction; In addition, picric acid, originally as yellow, is caught certain background colour can to the tissue after fixing, affect the experiment that subsequent embryo SABC and Hematoxylin-eosin dyeing (HE dyeing) etc. relate to color observation.Adopt picric acid concentration provided by the invention, coordinate other components, under the prerequisite ensureing picric effect, the background colour of fixing rear embryo can be reduced, be conducive to the observation of experimental result.
There is intermolecular cross-linking reaction in formaldehyde and albumen mass-energy, and through crosslinking, protein is fixed, in addition, formaldehyde can also preserve fat and lipoid, to chromosome, mitochondria and Golgi body, there is good fixation, but simple aldehyde fixative cannot stop the fixing follow-up dehydration of tissue, infiltration and embedding process to the extracting of sample lipid, thus it is undesirable to the fixed effect of film tissue in embryo, and its toxicity is large, generally needs to carry out in laboratory fume hood during operation.
Chromic acid is the good fixative of protein, also can help to preserve carbohydrate, but its penetration is lower, permeate slowlyer can not play for embryonic tissue the effect that fast shaping fixes.
Acetic acid can fix chromatin, and its dominant mechanism is that nucleic acid is precipitated, and histone dissolves, and stops chromosome condensation, makes chromosome structure avoid destroying; But be used alone and protoplasm can be made to expand, therefore easily make metaplasia, histocyte generation cracking, and damage overall sections observation effect.
The present invention adopts chromic acid, formaldehyde, picric acid and acetic acid to be made into certain density compound fixative, wherein, acetic acid can acetic acid permeability strong, there is intermolecular cross-linking reaction in formaldehyde and albumen mass-energy, the two can make the protein denaturation of cell membrane, strengthen the permeability of cell membrane, improve the fixing osmotic effect of fixative to embryonic tissue, thus contribute to the infiltration of picric acid and chromic acid.
Under each component of fixative has the prerequisite of good osmotic effect, formaldehyde in embryonic tissue fixative provided by the invention and picric acid are with the use of better to the embryonic tissue fixed effect being rich in film tissue, the hardness that embryonic tissue keeps suitable can be made, overcome acetic acid and be used alone the defect that can cause tissue bulking, make it to be suitable for slicing treatment, especially continuous wholely slicing treatment is carried out, the integrality of suitable hardness to embryonic tissue chip form has very large lifting, to the practicality that follow-up molecular pathology operates after strengthening section.
It is better that the concentration that formaldehyde in embryonic tissue fixative provided by the invention and chromic acid adopt penetrates effect to this material containing a large amount of lipopolysaccharide composition of embryo's film inner tissue, can overcome section and easily cause film defect and occur obtaining false positive or the inaccurate defect of testing result.And formaldehyde in embryonic tissue fixative provided by the invention and acetic acid, picric acid is with the use of effectively preserving chromosome.In addition, compared with the aldehyde fixative that Ma Er forint etc. is simple, the concentration of formaldehyde that the present invention adopts reduces greatly, reduces the toxicity of fixative.
In a word, each component of embryonic tissue fixative provided by the invention, by mutually coordinating, can prevent that embryonic tissue is molten to be split, and avoid tissue contracts, maintaining histogen has form; And it can permeate embryonic tissue fast and uniformly, and not easily make protoplasm expand, therefore can also fix by fast shaping.
Second aspect, the invention provides a kind of preparation method of embryonic tissue fixative as described in relation to the first aspect, gets chromate or potassium bichromate is water-soluble, then adds 2,4,6-trinitrophenol, formaldehyde and acetic acid, obtains described embryonic tissue fixative.
Preferably, described chromate is potassium chromate.
Preferably, described 2,4,6-trinitrophenols are for stating the saturated solution of 2,4,6-trinitrophenol, and its degree of saturation is 0.9% ~ 1.2%.
Preferably, described formaldehyde is the formaldehyde of for subsequent use 40%.
Preferably, described acetic acid is glacial acetic acid.
Preferably, the potassium bichromate solution of water-soluble obtained 12% mass fraction of described potassium bichromate is for subsequent use.
Preferably, the preparation method of embryonic tissue fixative provided by the invention is now with the current.
The preparation method of embryonic tissue fixative provided by the invention, preparation process is simple and quick, and after adding glacial acetic acid, itself and potassium bichromate effect generate chromic acid, and chromic acid is the good fixative of protein, improves the fixing osmotic effect to embryonic tissue.
In addition, the compound method of this embryonic tissue fixative is simple.
Preferably, embryonic tissue fixative provided by the invention is particularly useful for the fixing of mice embryonic.
The third aspect, the preparation method that the invention provides the embryonic tissue fixative that a kind of embryonic tissue fixative as described in relation to the first aspect or second party toilet are stated is preparing the application in histologic section, SABC, in situ hybridization or Hematoxylin-eosin dyeing (HE dyeing).
Preferably, histologic section is prepared for preparation mouse embryo tissue paraffin section.
Embryonic tissue fixative provided by the invention and its preparation method and application has following beneficial effect:
(1) embryonic tissue fixative provided by the invention can make embryonic tissue form intact, and the membranous structures such as chamber barrier film also can rapid osmotic, thus ensures effectively to detect great myocardium diseases such as Atrioventricular septums;
(2) adopt embryonic tissue fixative provided by the invention to obtain tissue morphology complete, and there is the section of suitable stiffness, thus can cut into slices continuous wholely, avoid section to occur cavitation phenomena, strengthen the practicality of follow-up molecular pathology operation;
(3) embryonic tissue fixative provided by the invention has higher protective efficacy for nucleic acid and albumen, destroys little to antigen, can be used for fixing to embryonic tissue in RT-PCR, in situ hybridization, SABC, immunofluorescence equimolecular pathological experiment;
(4) preparation method of the present invention is simple, with low cost.
Embodiment
The following stated is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Embodiment 1
A preparation method for embryonic tissue fixative, comprises the following steps:
Getting the water-soluble preparation quality mark of potassium bichromate is the potassium bichromate solution of 12%, add saturated 2 subsequently, 4,6-trinitrobenzen phenol solution (mass fraction about 1.22%), formaldehyde (volume fraction is 40%) and acetic acid, mixing, supply excess water, obtained embryonic tissue fixative, the percentage by weight of each component of described fixative is:
2,4,6-trinitrophenol 0.73%;
Formaldehyde 13.2%;
Chromic acid 0.48%;
Acetic acid 5.1%;
Surplus is water.
Embodiment 2
A preparation method for embryonic tissue fixative, comprises the following steps:
Get the potassium bichromate solution of the water-soluble preparation quality mark 12% of potassium bichromate, add saturated 2 subsequently, 4,6-trinitrobenzen phenol solution (mass fraction about 1.22%), formaldehyde (volume fraction is 40%) and acetic acid, mixing, supply excess water, obtained embryonic tissue fixative, the percentage by weight of each component of described fixative is:
2,4,6-trinitrophenol 0.67%;
Formaldehyde 11%;
Chromic acid 0.38%;
Acetic acid 4.2%;
Surplus is water.
Embodiment 3
A preparation method for embryonic tissue fixative, comprises the following steps:
Getting the water-soluble preparation quality mark of potassium bichromate is the potassium bichromate solution of 12%, add saturated 2 subsequently, 4,6-trinitrobenzen phenol solution (mass fraction about 1.22%), formaldehyde (volume fraction is 40%) and acetic acid, mixing, supply excess water, obtained embryonic tissue fixative, the percentage by weight of each component of described fixative is:
2,4,6-trinitrophenol 0.79%;
Formaldehyde 15.4%;
Chromic acid 0.58%;
Acetic acid 6.8%;
Surplus is water.
Embodiment 4
A preparation method for embryonic tissue fixative, comprises the following steps:
Getting the water-soluble preparation quality mark of potassium chromate is the potassium bichromate solution of 12%, add saturated 2 subsequently, 4,6-trinitrobenzen phenol solution (mass fraction about 1.22%), formaldehyde (volume fraction is 40%) and acetic acid, mixing, supply excess water, obtained embryonic tissue fixative, the percentage by weight of each component of described fixative is:
2,4,6-trinitrophenol 0.67%;
Formaldehyde 15.4%;
Chromic acid 0.58%;
Acetic acid 4.2%;
Surplus is water.
Embodiment 5
Adopt the obtained embryonic tissue fixative of embodiment 1 to be used for fixing of the conceived 14.5 days female mouse embryos in adult cleaning grade Kunming, comprise the following steps:
1. draw materials
Get the female mouse in healthy adult cleaning grade Kunming of conceived 14.5 days, cervical dislocation is put to death, and opens abdominal cavity, finds embryo, carefully separate embryo from the oviduct of both sides, uterus.
2. fixing
Wash by embryonic tissue physiological saline, drop in fixative fixing immediately, room temperature fixes 12-18h.
3. wash
Take out from fixative and organize immersion 50% ~ 70% ethanol 3 times, 5mim/ time.
4. dewater
30%, 50%, 70%, 80%, 90% ethanolic solution at different levels dewaters each 30min, puts into 95%, 100% each two sides three times, each 20min.
5. transparent
Immerse absolute alcohol, dimethylbenzene 1:1 equivalent mixed liquor: 15min, then put into dimethylbenzene 30min.
6. saturating wax
Put into dimethylbenzene, paraffin equivalent mixed liquor: 15min, then put into paraffin I, the paraffin II each 30min of wax thoroughly.
7. embed
By the tissue through saturating wax together with the paraffin of fusing, pour into together in container, then drop in cold water immediately, make it be frozen into wax stone at once, air drying saves backup.
8. paraffin section
1) paraffin mass platform wood that is fixing and that fix is contained on the insert platform of slicer.
2) be fixed on cutter holder by slicer, the edge of a knife upwards.
3) adjust the angle between paraffin mass and the edge of a knife and position, blade and paraffin section about become about 15 degree.
4) adjust caliper profiler to required slice thickness, be generally 4 ~ 10 microns.
5) section is started: this right hand shake runner, allow wax stone be cut into wax band, left hand is held writing brush and mentioned by wax band, and rocking-turn speed is generally 40 ~ 50r/min.
6) cutting wax disk(-sc) with single-edge blade a bit of, be placed on to carry on glass and add water one, being placed in magnifying glass or whether basis of microscopic observation section is good, as well then carried out paster.
7) paster
1. get the slide that a slice is clean, drip bonding die agent in slide central authorities, then smeared with clean finger, just uniformly thin layer.
2. the distilled water dripping 1 ~ 2 has been coated with on the slide of bonding die agent.
3. with pincet gripping in advance with the wax band that blade cuts open, be placed on the water surface, notice that the smooth one side of wax disk(-sc) light is affixed on slide, and make it to be in one end of slide slightly partially, the other end is convenient to adhesive label.
4. slide is set sheet position, be positioned on pre-heated exhibition sheet platform (temperature remains on 40 ~ 45 DEG C).Now wax disk(-sc) stretches because being heated and shakeouts.
5. open up slide to be placed on after sheet and square position finishes mark and be placed in 37 DEG C of incubators and dry, can take out after diel drying and deposit in section box.
9. gained section is used for SABC, in situ hybridization, HE dyeing equimolecular pathology, structure observation experiment.
" h " expression hour in the present invention, " min " expression minute.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. an embryonic tissue fixative, is characterized in that, this embryonic tissue fixative contains 2,4,6-trinitrophenol, formaldehyde, chromic acid and acetic acid;
The percentage by weight of each component of described embryonic tissue fixative is:
2,4,6-trinitrophenol 0.67% ~ 0.79%;
Formaldehyde 11% ~ 15.4%;
Chromic acid 0.38% ~ 0.58%;
Acetic acid 4.2% ~ 6.8%;
Surplus is water.
2. embryonic tissue fixative as claimed in claim 1, it is characterized in that, the percentage by weight of each component of described embryonic tissue fixative is:
2,4,6-trinitrophenol 0.73%;
Formaldehyde 13.2%;
Chromic acid 0.48%;
Acetic acid 5.1%;
Surplus is water.
3. the preparation method of embryonic tissue fixative as claimed in claim 1, is characterized in that, gets chromate or potassium bichromate is water-soluble, then adds 2,4,6-trinitrophenol, formaldehyde and acetic acid, obtains described embryonic tissue fixative.
4. the application in histologic section, SABC, in situ hybridization or Hematoxylin-eosin dyeing prepared by embryonic tissue fixative as claimed in claim 1.
5. the application of embryonic tissue fixative as claimed in claim 1 in preparation mouse embryo tissue paraffin section.
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Application publication date: 20150603 |