CN105699142A - Paraffin section making method for close-texture hard plant materials - Google Patents
Paraffin section making method for close-texture hard plant materials Download PDFInfo
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- CN105699142A CN105699142A CN201610090674.4A CN201610090674A CN105699142A CN 105699142 A CN105699142 A CN 105699142A CN 201610090674 A CN201610090674 A CN 201610090674A CN 105699142 A CN105699142 A CN 105699142A
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
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- G01N1/36—Embedding or analogous mounting of samples
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Abstract
The invention relates to the technical field of plant sample tissue paraffin sections, in particular to a paraffin section making method for grain of close-texture hard plant materials such as rice and wheat at the late development stage. The paraffin section making method includes the following steps of: (1) fixation, namely subjecting plant seed materials at different stages after pollination to longitudinal cutting or cross cutting, and putting the cut pieces into fixing solution for fixing at room temperature for 3-5h; (2) dehydration, namely using a pipette for sucking out the fixing solution, and sequentially adding 30%, 50%, 75%, 95% and 100% alcohol in gradient for dehydration; (3) transparency, namely after using the pipette to suck out the alcohol, using chloroform as a transparent agent; (4) paraffin impregnation; (5) embedding and sectioning. The method is high in operability, simple, economical and practical, the problem that the sections of grains at the late development stage are fragile can be solved, and complete paraffin sections of the grains at the late development stage can be cut out easily.
Description
Technical field
The present invention relates to the vegetable materials such as plant sample tissue paraffin section de technical field, especially a kind of rice wheat closely hard for quality, such as the paraffin section manufacture method of Wheat Development later stage seed。
Background technology
Paraffin section technology is to be engaged in the basic test technical method of developmental biology and Histochemical studies, is also microexamination organization structure of the plant common technology。This technology of botany research field is built up in research phytomorph, extensive application in plant hybridization breeding, the cultivation of rare plant and discriminating etc.。In addition, histochemical stain is carried out for animal and plant sample, carry out immunohistochemical study and carries out the researchs such as in situ hybridization, all it be unable to do without the making that animal and plant sample tissue is carried out paraffin section, and the excellent Degree of Accord Relation that makes of paraffin section is to the order of accuarcy of correlational study result of the test。Therefore, paraffin section technology is the basic test technology carrying out animal and plant developmental biology and Histochemical studies at life science。
Wheat seed is grown and is started substantially experience kernel grouting initial stage, milk stage, stage of wax ripeness, the stage of yellow ripeness and full ripe stage from chasmogamy。Along with kernel grouting process continues, wheat seed form being changed from small to big, along with in seed, starch and albumen accumulate gradually, wheat seed is developed into the tight hard solid state endosperm of quality gradually by the liquid endosperm of Early filling stage。Manufacture method for seed paraffin sections such as rice wheats, (Kang Haiqi etc. by literature search, 2010) method inquired is according to conventional plant paraffin microsection manufacture universal method mostly, and has reported that the paraffin section method that correlational study uses can only cut out grouting early stage seed paraffin section。Due to wheat seed Early filling stage, relatively soft being easier to of kernel texture cuts out complete paraffin section, and wheat seed development later stage kernel texture is closely hard, frangible during section more difficult cuts out entire kernel paraffin section。The main cause of Wheat Development later stage seed section difficulty has two aspects: on the one hand from seed material, owing to wheat seed grows a large amount of accumulation and the moisture content of kernels reduction of middle and late stage starch and protein, causing seed material hardening, endosperm tissue quality becomes fine and close。On the other hand for dicing method, traditional paraffin section preparation methods is summarised as dehydration of alcohol, and dimethylbenzene is transparent, then the method for waxdip step by step。Owing to dimethylbenzene can make organization material be contracted thereby to become hard, thus resulting in this just fine and close seed material waxdip can be more difficult, and the traditional paraffin section preparation methods waxdip time is too short, this results in paraffin and is not completely immersed in inside seed histiocyte, therefore when doing seed section, material is extremely fragile, is difficult to cut out the paraffin section of entire kernel。(2010) have just been waited to attempt seed material is carried out sofening treatment dawn on rice grain paraffin section makes, namely 1/2 glycerol+1/2 dehydrated alcohol mixing replaces dimethylbenzene, clearing time is one week, result seed can become transparent, but not easily cut into slices, its method cannot cut out the entire kernel paraffin section of more than 12d after pollination。Kang Haiqi etc. use the hydrofluoric acid solution softening seed of 0.5%～15%, and the time is from 30min to 30d, but material is still not easily cut into slices；If being boiled by rice so as to soften, endosperm starch will become gelatinizing shape, and this method also can only be switched to the seed of 11d after pollination。
Therefore, Wheat Development later stage seed section complete paraffin section frangible, that research worker is not easy to cut out Wheat Development later stage seed is a problem anxious to be resolved。
Summary of the invention
It is an object of the invention to provide a kind of workable, method is easy, economical and practical, can solve the problem that the development later stage seed frangible problem of section, it is easy to cut out the paraffin section manufacture method for the tight hard vegetable material of quality of the complete paraffin section of development later stage seed。
The invention discloses a kind of paraffin section manufacture method for the tight hard vegetable material of quality, it is characterised in that comprise the following steps:
(1), fixing:
After taking pollination, the plant seed material of different times carries out rip cutting or crosscut, puts in the fixative in container, uses vacuum desiccator evacuation, all sinks to container bottom to all seed materials, and room temperature fixes 3～5h；
This step points for attention:
(1), be the chloroform that can corrode plastics owing to making the clarifier that the section later stage uses, thus start to use vial from material is fixing, and plastic centrifuge tube should not be used。
(2), applicable fixative should being selected according to subsequent experimental specific requirement, if making the paraffin section in situ hybridization, needing to use (4% paraformaldehyde+0.1%(v/v) DEPC) as fixative composition。It addition, need to select suitable fixative according to experiment, such as make the paraffin section in situ hybridization, 4% paraformaldehyde need to be used to fix, and add 0.1%(v:v) DEPC；Such as the observation of plant histiocyte that need to dye, and plant tissue need to be preserved for a long time, it is possible to use FAA fixative；As DAPI dyeing (DAPI is a kind of fluorescent dye, it is possible to specifically DNA is dyeed, under fluorescence can nucleus in tissues observed) need to be carried out, Kano fixative can be used。
(3), seed must carry out rip cutting or crosscut, otherwise fixative is difficult to enter, and can cause that seed is outside excessively fixing and inside is not also fixed, simultaneously the steps such as also dehydration after impact is transparent。
(2), dehydration:
Utilize liquid-transfering gun by above-mentioned fixative sucking-off, be successively sequentially added into the ethanol that gradient is 30%, 50%, 75%, 95% and 100% concentration and carry out dehydration: 30% dehydration of alcohol 3～5 times, change an ethanol every 20～30min；50% dehydration of alcohol 3～5 times, changes an ethanol every 20～30min；Afterwards sample it is transferred on shaking table or shakes container every 8～12min, 75% dehydration of alcohol 3～5 times, change an ethanol every 40～50min；95% dehydration of alcohol 3～5 times, changes an ethanol every 40～50min；100% absolute alcohol dehydration 3～5 times, changes an ethanol every 40～50min；
This step points for attention:
(1) it appeared that the chlorophyll in Early filling stage seed fruit seed coat gradually by alcoholic extract out, turns green near the ethanol at the bottom of bottle, therefore uses shaking table to rock, to keep ethanol uniform, make seed dehydration be smoothed out after, changing 75% ethanol。
(2), this step can not also use shaking table, but need to shake container every about 10min。Chlorophyll has not been had to be extracted out during to the last ethanol of replacing for several times, it was shown that seed dehydration is complete。
Conventional method often walks dewatering time and is generally 1～2h, and this step extends dewatering time compared with conventional method, uses shaking table to make dehydration more smooth and easy simultaneously, and easily determines whether seed dehydration completes。
(3), transparent:
Utilizing imbibition rifle after above-mentioned ethanol sucking-off, chloroform will to be used to make clarifier, addition volume ratio is ethanol: chloroform=3:1 solution left standstill 3～4h, is evacuated to all material and sinks to the bottom；Utilizing imbibition rifle by solution sucking-off afterwards, addition volume ratio is ethanol: chloroform=1:1 solution, stands 3～4h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, addition volume ratio is ethanol: chloroform=1:3 solution, stands 3～4h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, add pure chloroform and stand 2～3d, be evacuated to all material and sink to the bottom；
This step points for attention:
(1), chloroform Material shrinkage will not be made hardening, therefore use chloroform give clarifier instead。But chloroform seepage velocity is relatively slow, due to wheat seed grouting, later stage material self is harder, water content is relatively low, and therefore this step clearing time to be grown more than conventional process time。
(2), owing to chloroform density is relatively big, when improving chloroform ratio, material all can be floated to liquid level every time, so need evacuation to promote that clarifier enters inside material structure every time。
(3) when, transparent step terminates, it is possible to find that the grouting later stage material of (such as 35d after pollination) can also printing opacity facing to light source, it was shown that this step of seed is transparent well。
This step is used chloroform instead and is made clarifier, and significantly extends clearing time, it is possible to accomplish not make Material shrinkage hardening and transparent completely。
(4), waxdip:
Utilizing imbibition rifle by after described above-mentioned pure chloroform sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=3:1, is incubated 4～5h at 40～42 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: paraffin=1:1, is incubated 4～5h at 40～42 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=1:3, is incubated 4～5h at 45～47 DEG C；For the material before 21d after pollination, paraffin refined wax is utilized to be incubated 3～4d at 55～57 DEG C；For material later for 21d after pollination, utilize paraffin refined wax to be incubated 3～5d at 55～57 DEG C, during waxdip, change a paraffin refined wax；
This step points for attention:
(1), the starch of wheat seed own and protein content higher, especially arrive the Grain Development later stage all the more so, cause that waxdip is very difficult。Therefore the waxdip time must be extended, and suitable temperature during waxdip, must be kept, the too high structure not only broken up within seed of temperature, and during higher than 60 DEG C, it is unfavorable for preserving the bioactive substance of organization internal, affect the result of SABC, also can make that tissue is hardening, become fragile, also result in seed entirety loose, disintegrate, therefore, keep 55 DEG C of waxdips, make paraffin be in slush state。
(2), waxdip completely after, it is possible to find that material becomes micro-green or white translucent bowlder-like。
This step keeps relatively low waxdip temperature, but significantly extends the waxdip time, it is possible to makes complete waxdip in seed histiocyte, can ensure that material completely and not destroys internal structure, moreover it is possible to preserve the bioactive substance of organization internal simultaneously。
As shown in Figure 2, it it is the wheat seed cross section comparison diagram after the inventive method and traditional method waxdip, in figure, A, B are expressed as the effect after Semen Tritici aestivi spends the seed of rear 21d and 28d to use this method waxdip, and the effect after the seed use traditional method waxdip of rear 21d and 28d spent by C, D respectively Semen Tritici aestivi。As in figure 2 it is shown, use the wheat seed waxdip after this method waxdip complete, be translucent shape, does not have starch and drops, and after using traditional method waxdip, wheat seed waxdip is insufficient, has starch and drop during section during section。
(5), embedding and section:
The seed material carton used or paraffin embedding box embed according to a conventional method, can directly cut into slices on paraffin slicing machine according to a conventional method for the material before 21d after pollination, blade must be used after material paraffin embedding later for 21d after pollination to cut out material cross-section, then by wax stone back-off in DEPC water 4～6 DEG C softening overnight, then cutting into slices, above-mentioned all seed materials all can be cut into the section of thickness range 8～25 μm again。
This step adds DEPC water softening step for Grain Development later stage material, makes the seed that the Grain Development later stage has been mature on the whole also can cut out whole slices。
The paraffin section made through the method is after roasting sheet, dewaxing and rehydration, it is possible to according to follow-up study experiment purpose, enter ensuing experimental implementation。
As shown in Figure 3, it it is the contrast picture of the wheat seed paraffin section utilizing the inventive method and traditional method to cut out, shown in figure: A, B, C respectively Semen Tritici aestivi spend after 21,28, the seed of the 35d paraffin section that uses this method to cut out, D, E, F respectively Semen Tritici aestivi spend after 21,28, the seed of the 35d paraffin section that uses traditional method to cut out。
As it is shown on figure 3, use the inventive method can cut out the paraffin section that wheat seed development later stage is complete, and use the paraffin section that traditional method cuts out substantially all imperfect。
The basic functional principle of the present invention is:
Paraffin section technology is to do one of life science common technology, owing to starch and protein accumulation, in addition moisture content of kernels reduce in the development of plants later stage seeds such as Semen Tritici aestivi, seed material can be caused hardening, and grain endosperm tissue becomes fine and close。Using traditional paraffin section preparation methods that seed Material shrinkage can be made hardening, material waxdip is very difficult, thus very easily broken during section, is difficult to cut out complete paraffin section。For solving this problem, the present invention, through repeatedly attempting and improving, changes clarifier, extends clearing time, adjust waxdip time and temperature, it is proposed to make new method for the tight hard vegetable material of quality (Wheat Development later stage seed) paraffin section。Application this method can make wheat seed from Early filling stage to seed complete ripeness during the entire kernel paraffin section in each period, it is possible to be applied in wheat seed developmental biology and histochemical correlational study。
By the improvement of constantly bringing forth new ideas to Wheat Development later stage seed paraffin section method, the present invention is directed to the characteristic that Wheat Development later stage kernel texture is closely hard, it is proposed by adopting chloroform as clarifier, and extend clearing time to 3d, and adjust waxdip time and temperature, paraffin can be made to be completely immersed in seed organization internal, solve the Wheat Development later stage seed frangible problem of section, it is possible to make research worker be easy to cut out the complete paraffin section of Wheat Development later stage seed。
As shown in Figure 4, for wheat seed different times paraffin section through I2-KI dye after microphotograph, in figure A, B, C, D respectively Semen Tritici aestivi spend after 14,21,28, the microphotograph taken pictures immediately after I2-KI dyes of the seed paraffin section of 35d, E, F, G, H respectively Semen Tritici aestivi spend after 14,21,28, the microphotograph taken pictures after I2-KI dyes 48h of the seed paraffin section of 35d。The inventive method is used to produce the paraffin section that after wheat seed is pollinated, each period is complete。As Fig. 4 be the section using the method to make take pictures immediately (in figure A～D) and dye after I2-KI dyes (in figure E～H) after 48h take pictures the microphotograph obtained。The distribution (in figure A～D) of starch and cellularity (in figure E～H) clearly from figure it will be clear that in wheat seed albuminous cell。
Compared with prior art, the present invention be a kind of workable, method is easy, economical and practical, can solve the problem that the development later stage seed frangible problem of section, it is easy to cut out the paraffin section manufacture method for the tight hard vegetable material of quality of the complete paraffin section of development later stage seed。
Accompanying drawing explanation
Fig. 1 is the invention process flow chart of steps。
Fig. 2 is be the inventive method with traditional method waxdip after wheat seed cross section comparison diagram, in figure, A, B are expressed as the effect after Semen Tritici aestivi spends the seed of rear 21d and 28d to use this method waxdip, and the effect after the seed use traditional method waxdip of rear 21d and 28d spent by C, D respectively Semen Tritici aestivi。
Fig. 3 is the contrast picture of the wheat seed paraffin section utilizing the inventive method and traditional method to cut out, shown in figure: A, B, C respectively Semen Tritici aestivi spend after 21,28, the seed of the 35d paraffin section that uses this method to cut out, D, E, F respectively Semen Tritici aestivi spend after 21,28, the seed of the 35d paraffin section that uses traditional method to cut out。
Fig. 4 is wheat seed different times paraffin section microphotograph after I2-KI dyes, in figure A, B, C, D respectively Semen Tritici aestivi spend after 14,21,28, the microphotograph taken pictures immediately after I2-KI dyes of the seed paraffin section of 35d, E, F, G, H respectively Semen Tritici aestivi spend after 14,21,28, the microphotograph taken pictures after I2-KI dyes 48h of the seed paraffin section of 35d。
Detailed description of the invention
Embodiment 1:
Taking each 5 of the fresh wheat seed of 7,14,21,28,35d after pollination respectively, put into vial after crosscut, add 5ml4% paraformaldehyde fixative, evacuation fixes seed。Next operate according to this method, wheat seed can be cut out and grow complete paraffin section in each period。
With reference to Fig. 1-Fig. 4, a kind of paraffin section manufacture method for the tight hard vegetable material of quality, comprise the following steps:
(1), fixing:
Taking each 5 of the fresh wheat seed of 7,14,21,28,35d after pollination respectively, put into vial after crosscut, add 5ml4% paraformaldehyde fixative, use vacuum desiccator evacuation, all sink to container bottom to all seed materials, room temperature fixes 4h；
This step points for attention:
(1), be the chloroform that can corrode plastics owing to making the clarifier that the section later stage uses, thus start to use vial from material is fixing, and plastic centrifuge tube should not be used。
(2), applicable fixative should being selected according to subsequent experimental specific requirement, if making the paraffin section in situ hybridization, needing to use (4% paraformaldehyde+0.1%(v/v) DEPC) as fixative composition。It addition, need to select suitable fixative according to experiment, such as make the paraffin section in situ hybridization, 4% paraformaldehyde need to be used to fix, and add 0.1%(v:v) DEPC；Such as the observation of plant histiocyte that need to dye, and plant tissue need to be preserved for a long time, it is possible to use FAA fixative；As DAPI dyeing (DAPI is a kind of fluorescent dye, it is possible to specifically DNA is dyeed, under fluorescence can nucleus in tissues observed) need to be carried out, Kano fixative can be used。
(3), seed must carry out rip cutting or crosscut, otherwise fixative is difficult to enter, and can cause that seed is outside excessively fixing and inside is not also fixed, simultaneously the steps such as also dehydration after impact is transparent。
(2), dehydration:
Utilize liquid-transfering gun by above-mentioned fixative sucking-off, be successively sequentially added into the ethanol that gradient is 30%, 50%, 75%, 95% and 100% concentration and carry out dehydration: 30% dehydration of alcohol 4 times, change an ethanol every 30min；50% dehydration of alcohol 4 times, changes an ethanol every 30min；Afterwards sample it is transferred on shaking table or shakes container every 8min, 75% dehydration of alcohol 4 times, change an ethanol every 40min；95% dehydration of alcohol 4 times, changes an ethanol every 40min；100% absolute alcohol dehydration 4 times, changes an ethanol every 40min；
This step points for attention:
(1) it appeared that the chlorophyll in Early filling stage seed fruit seed coat gradually by alcoholic extract out, turns green near the ethanol at the bottom of bottle, therefore uses shaking table to rock, to keep ethanol uniform, make seed dehydration be smoothed out after, changing 75% ethanol。
(2), this step can not also use shaking table, but need to shake container every about 10min。Chlorophyll has not been had to be extracted out during to the last ethanol of replacing for several times, it was shown that seed dehydration is complete。
Conventional method often walks dewatering time and is generally 1～2h, and this step extends dewatering time compared with conventional method, uses shaking table to make dehydration more smooth and easy simultaneously, and easily determines whether seed dehydration completes。
(3), transparent:
Utilizing imbibition rifle after above-mentioned ethanol sucking-off, chloroform will to be used to make clarifier, addition volume ratio is ethanol: chloroform=3:1 solution left standstill 3h, is evacuated to all material and sinks to the bottom；Utilizing imbibition rifle by solution sucking-off afterwards, addition volume ratio is ethanol: chloroform=1:1 solution, stands 3h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, addition volume ratio is ethanol: chloroform=1:3 solution, stands 3h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, add pure chloroform and stand 3d, be evacuated to all material and sink to the bottom；
This step points for attention:
(1), chloroform Material shrinkage will not be made hardening, therefore use chloroform give clarifier instead。But chloroform seepage velocity is relatively slow, due to wheat seed grouting, later stage material self is harder, water content is relatively low, and therefore this step clearing time to be grown more than conventional process time。
(2), owing to chloroform density is relatively big, when improving chloroform ratio, material all can be floated to liquid level every time, so need evacuation to promote that clarifier enters inside material structure every time。
(3) when, transparent step terminates, it is possible to find that the grouting later stage material of (such as 35d after pollination) can also printing opacity facing to light source, it was shown that this step of seed is transparent well。
This step is used chloroform instead and is made clarifier, and significantly extends clearing time, it is possible to accomplish not make Material shrinkage hardening and transparent completely。
(4), waxdip:
Utilizing imbibition rifle by after described above-mentioned pure chloroform sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=3:1, is incubated 4h at 40 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: paraffin=1:1, is incubated 4h at 40 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=1:3, is incubated 4h at 45 DEG C；For the material before 21d after pollination, paraffin refined wax is utilized to be incubated 3d at 55 DEG C；For material later for 21d after pollination, utilize paraffin refined wax to be incubated 3d at 55 DEG C, during waxdip, change a paraffin refined wax；
This step points for attention:
(1), the starch of wheat seed own and protein content higher, especially arrive the Grain Development later stage all the more so, cause that waxdip is very difficult。Therefore the waxdip time must be extended, and suitable temperature during waxdip, must be kept, the too high structure not only broken up within seed of temperature, and during higher than 60 DEG C, it is unfavorable for preserving the bioactive substance of organization internal, affect the result of SABC, also can make that tissue is hardening, become fragile, also result in seed entirety loose, disintegrate, therefore, keep 55 DEG C of waxdips, make paraffin be in slush state。
(2), waxdip completely after, it is possible to find that material becomes micro-green or white translucent bowlder-like。
This step keeps relatively low waxdip temperature, but significantly extends the waxdip time, it is possible to makes complete waxdip in seed histiocyte, can ensure that material completely and not destroys internal structure, moreover it is possible to preserve the bioactive substance of organization internal simultaneously。
As shown in Figure 2, it it is the wheat seed cross section comparison diagram after the inventive method and traditional method waxdip, in figure, A, B are expressed as the effect after Semen Tritici aestivi spends the seed of rear 21d and 28d to use this method waxdip, and the effect after the seed use traditional method waxdip of rear 21d and 28d spent by C, D respectively Semen Tritici aestivi。As in figure 2 it is shown, use the wheat seed waxdip after this method waxdip complete, be translucent shape, does not have starch and drops, and after using traditional method waxdip, wheat seed waxdip is insufficient, has starch and drop during section during section。
(5), embedding and section:
The seed material carton used or paraffin embedding box embed according to a conventional method, can directly cut into slices on paraffin slicing machine according to a conventional method for the material before 21d after pollination, blade must be used after material paraffin embedding later for 21d after pollination to cut out material cross-section, then by wax stone back-off in DEPC water 4 DEG C softening overnight, then cutting into slices, above-mentioned all seed materials all can be cut into the section of thickness range 8 μm again。
This step adds DEPC water softening step for Grain Development later stage material, makes the seed that the Grain Development later stage has been mature on the whole also can cut out whole slices。
The paraffin section made through the method is after roasting sheet, dewaxing and rehydration, it is possible to according to follow-up study experiment purpose, enter ensuing experimental implementation。
As shown in Figure 3, it it is the contrast picture of the wheat seed paraffin section utilizing the inventive method and traditional method to cut out, shown in figure: A, B, C respectively Semen Tritici aestivi spend after 21,28, the seed of the 35d paraffin section that uses this method to cut out, D, E, F respectively Semen Tritici aestivi spend after 21,28, the seed of the 35d paraffin section that uses traditional method to cut out。
As it is shown on figure 3, use the inventive method can cut out the paraffin section that wheat seed development later stage is complete, and use the paraffin section that traditional method cuts out substantially all imperfect。
As shown in Figure 4, for wheat seed different times paraffin section through I2-KI dye after microphotograph, in figure A, B, C, D respectively Semen Tritici aestivi spend after 14,21,28, the microphotograph taken pictures immediately after I2-KI dyes of the seed paraffin section of 35d, E, F, G, H respectively Semen Tritici aestivi spend after 14,21,28, the microphotograph taken pictures after I2-KI dyes 48h of the seed paraffin section of 35d。The inventive method is used to produce the paraffin section that after wheat seed is pollinated, each period is complete。As Fig. 4 be the section using the method to make take pictures immediately (in figure A～D) and dye after I2-KI dyes (in figure E～H) after 48h take pictures the microphotograph obtained。The distribution (in figure A～D) of starch and cellularity (in figure E～H) clearly from figure it will be clear that in wheat seed albuminous cell。
Embodiment 2:
A kind of paraffin section manufacture method for the tight hard vegetable material of quality, comprises the following steps:
(1), fixing:
Taking each 5 of the fresh wheat seed of 7,14,21,28,35d after pollination respectively, put into vial after crosscut, add 5ml4% paraformaldehyde fixative, use vacuum desiccator evacuation, all sink to container bottom to all seed materials, room temperature fixes 3h；
(2), dehydration:
Utilize liquid-transfering gun by above-mentioned fixative sucking-off, be successively sequentially added into the ethanol that gradient is 30%, 50%, 75%, 95% and 100% concentration and carry out dehydration: 30% dehydration of alcohol 3 times, change an ethanol every 25min；50% dehydration of alcohol 3 times, changes an ethanol every 20min；Afterwards sample it is transferred on shaking table or shakes container every 10min, 75% dehydration of alcohol 5 times, change an ethanol every 45min；95% dehydration of alcohol 5 times, changes an ethanol every 45min；100% absolute alcohol dehydration 3 times, changes an ethanol every 45min；
(3), transparent:
Utilizing imbibition rifle after above-mentioned ethanol sucking-off, chloroform will to be used to make clarifier, addition volume ratio is ethanol: chloroform=3:1 solution left standstill 4h, is evacuated to all material and sinks to the bottom；Utilizing imbibition rifle by solution sucking-off afterwards, addition volume ratio is ethanol: chloroform=1:1 solution, stands 3.5h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, addition volume ratio is ethanol: chloroform=1:3 solution, stands 3.5h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, add pure chloroform and stand 2d, be evacuated to all material and sink to the bottom；
(4), waxdip:
Utilizing imbibition rifle by after described above-mentioned pure chloroform sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=3:1, is incubated 4.5h at 41 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: paraffin=1:1, is incubated 4.5h at 41 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=1:3, is incubated 4.5h at 46 DEG C；For the material before 21d after pollination, paraffin refined wax is utilized to be incubated 3.5d at 56 DEG C；For material later for 21d after pollination, utilize paraffin refined wax to be incubated 4d at 56 DEG C, during waxdip, change a paraffin refined wax；
(5), embedding and section:
The seed material carton used or paraffin embedding box embed according to a conventional method, can directly cut into slices on paraffin slicing machine according to a conventional method for the material before 21d after pollination, blade must be used after material paraffin embedding later for 21d after pollination to cut out material cross-section, then by wax stone back-off in DEPC water 5 DEG C softening overnight, then cutting into slices, above-mentioned all seed materials all can be cut into the section of thickness range 20 μm again。
Embodiment 3:
A kind of paraffin section manufacture method for the tight hard vegetable material of quality, comprises the following steps:
(1), fixing:
Taking each 5 of the fresh wheat seed of 7,14,21,28,35d after pollination respectively, put into vial after crosscut, add 5ml4% paraformaldehyde fixative, use vacuum desiccator evacuation, all sink to container bottom to all seed materials, room temperature fixes 5h；
(2), dehydration:
Utilize liquid-transfering gun by above-mentioned fixative sucking-off, be successively sequentially added into the ethanol that gradient is 30%, 50%, 75%, 95% and 100% concentration and carry out dehydration: 30% dehydration of alcohol 5 times, change an ethanol every 20min；50% dehydration of alcohol 5 times, changes an ethanol every 25min；Afterwards sample it is transferred on shaking table or shakes container every 12min, 75% dehydration of alcohol 3 times, change an ethanol every 50min；95% dehydration of alcohol 3 times, changes an ethanol every 50min；100% absolute alcohol dehydration 5 times, changes an ethanol every 50min；
(3), transparent:
Utilizing imbibition rifle after above-mentioned ethanol sucking-off, chloroform will to be used to make clarifier, addition volume ratio is ethanol: chloroform=3:1 solution left standstill 3.5h, is evacuated to all material and sinks to the bottom；Utilizing imbibition rifle by solution sucking-off afterwards, addition volume ratio is ethanol: chloroform=1:1 solution, stands 4h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, addition volume ratio is ethanol: chloroform=1:3 solution, stands 4h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, add pure chloroform and stand 2.5d, be evacuated to all material and sink to the bottom；
(4), waxdip:
Utilizing imbibition rifle by after described above-mentioned pure chloroform sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=3:1, is incubated 5h at 42 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: paraffin=1:1, is incubated 5h at 42 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=1:3, is incubated 5h at 47 DEG C；For the material before 21d after pollination, paraffin refined wax is utilized to be incubated 4d at 57 DEG C；For material later for 21d after pollination, utilize paraffin refined wax to be incubated 5d at 57 DEG C, during waxdip, change a paraffin refined wax；
(5), embedding and section:
The seed material carton used or paraffin embedding box embed according to a conventional method, can directly cut into slices on paraffin slicing machine according to a conventional method for the material before 21d after pollination, blade must be used after material paraffin embedding later for 21d after pollination to cut out material cross-section, then by wax stone back-off in DEPC water 6 DEG C softening overnight, then cutting into slices, above-mentioned all seed materials all can be cut into the section of thickness range 25 μm again。
Claims (1)
1. the paraffin section manufacture method for the tight hard vegetable material of quality, it is characterised in that comprise the following steps:
(1), fixing:
After taking pollination, the plant seed material of different times carries out rip cutting or crosscut, puts in the fixative in container, uses vacuum desiccator evacuation, all sinks to container bottom to all seed materials, and room temperature fixes 3～5h；
(2), dehydration:
Utilize liquid-transfering gun by above-mentioned fixative sucking-off, be successively sequentially added into the ethanol that gradient is 30%, 50%, 75%, 95% and 100% concentration and carry out dehydration: 30% dehydration of alcohol 3～5 times, change an ethanol every 20～30min；50% dehydration of alcohol 3～5 times, changes an ethanol every 20～30min；Afterwards sample it is transferred on shaking table or shakes container every 8～12min, 75% dehydration of alcohol 3～5 times, change an ethanol every 40～50min；95% dehydration of alcohol 3～5 times, changes an ethanol every 40～50min；100% absolute alcohol dehydration 3～5 times, changes an ethanol every 40～50min；
(3), transparent:
Utilizing imbibition rifle after above-mentioned ethanol sucking-off, chloroform will to be used to make clarifier, addition volume ratio is ethanol: chloroform=3:1 solution left standstill 3～4h, is evacuated to all material and sinks to the bottom；Utilizing imbibition rifle by solution sucking-off afterwards, addition volume ratio is ethanol: chloroform=1:1 solution, stands 3～4h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, addition volume ratio is ethanol: chloroform=1:3 solution, stands 3～4h, is evacuated to all material and sinks to the bottom；Again with imbibition rifle by solution sucking-off, add pure chloroform and stand 2～3d, be evacuated to all material and sink to the bottom；
(4), waxdip:
Utilizing imbibition rifle by after described above-mentioned pure chloroform sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=3:1, is incubated 4～5h at 40～42 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: paraffin=1:1, is incubated 4～5h at 40～42 DEG C；Afterwards, utilizing imbibition rifle by after above-mentioned chloroform and paraffin mixed liquor sucking-off, addition volume ratio is chloroform: the mixed liquor of paraffin=1:3, is incubated 4～5h at 45～47 DEG C；For the material before 21d after pollination, paraffin refined wax is utilized to be incubated 3～4d at 55～57 DEG C；For material later for 21d after pollination, utilize paraffin refined wax to be incubated 3～5d at 55～57 DEG C, during waxdip, change a paraffin refined wax；
(5), embedding and section:
The seed material carton used or paraffin embedding box embed according to a conventional method, can directly cut into slices on paraffin slicing machine according to a conventional method for the material before 21d after pollination, blade must be used after material paraffin embedding later for 21d after pollination to cut out material cross-section, then by wax stone back-off in DEPC water 4～6 DEG C softening overnight, then cutting into slices, above-mentioned all seed materials all can be cut into the section of thickness range 8～25 μm again。
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053177A (en) * | 2016-07-28 | 2016-10-26 | 江苏农林职业技术学院 | Specimen making method for observing anatomic structure of lamina of tillandsia |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102967493A (en) * | 2012-10-27 | 2013-03-13 | 山西农业大学 | Rapid paraffin sectioning method for plant tissue |
| CN103234797A (en) * | 2013-04-28 | 2013-08-07 | 中南大学湘雅三医院 | Dehydrating and embedding improvement method of human body or animal tissues |
| CN103305600A (en) * | 2013-01-25 | 2013-09-18 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof |
| WO2015034178A1 (en) * | 2013-09-09 | 2015-03-12 | 국립암센터 | Blood cell aggregating agent for preparing paraffin block and method for preparing paraffin block by using same |
| JP2015114139A (en) * | 2013-12-10 | 2015-06-22 | 村角工業株式会社 | Tray for preparing paraffin block |
-
2016
- 2016-02-18 CN CN201610090674.4A patent/CN105699142B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102967493A (en) * | 2012-10-27 | 2013-03-13 | 山西农业大学 | Rapid paraffin sectioning method for plant tissue |
| CN103305600A (en) * | 2013-01-25 | 2013-09-18 | 海尔施生物医药股份有限公司 | Kit for synchronously detecting related gene expression level of 14 antitumor drugs by using paraffin embedding biopsy sample, and detection method thereof |
| CN103234797A (en) * | 2013-04-28 | 2013-08-07 | 中南大学湘雅三医院 | Dehydrating and embedding improvement method of human body or animal tissues |
| WO2015034178A1 (en) * | 2013-09-09 | 2015-03-12 | 국립암센터 | Blood cell aggregating agent for preparing paraffin block and method for preparing paraffin block by using same |
| JP2015114139A (en) * | 2013-12-10 | 2015-06-22 | 村角工業株式会社 | Tray for preparing paraffin block |
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