CN102607930A - Fish germ cell orientated embedding technology - Google Patents
Fish germ cell orientated embedding technology Download PDFInfo
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- CN102607930A CN102607930A CN2012100651130A CN201210065113A CN102607930A CN 102607930 A CN102607930 A CN 102607930A CN 2012100651130 A CN2012100651130 A CN 2012100651130A CN 201210065113 A CN201210065113 A CN 201210065113A CN 102607930 A CN102607930 A CN 102607930A
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Abstract
The invention discloses a fish germ cell orientated embedding technology, relating to an orientated embedding technology and solving the problems of easy loss of fertilized parts and poor cutting quality in the traditional tabletting process. The method I comprises the steps of: carrying out dewatering, clearing and wax absorbing on fish germs after fixing; carrying out locating and wax trimming after embedding; labeling the fertilized parts and cutting, and carrying out secondary orientated embedding to prepare a light microscope embedding block; and preparing a conventional light microscope. The method II comprises the steps of: carrying out dewatering, clearing and wax absorbing on fish germs after fixing; carrying out locating and wax trimming after embedding; labeling the fertilized parts and cutting, carrying out secondary fixing, back-washing and dewatering, and carrying out orientated embedding and polymerizing on epoxy resin #618 to obtain an electron microscope embedding block; and preparing a conventional electron microscope. A sample of the light microscope or electron microscope can ensure that fish germs can be completely cut and required contents can be accurately observed from the fertilized parts of the fish germs, and has the advantages of good cutting quality, clear image and better observation effect. Meanwhile, according to the fish germ cell orientated embedding technology, a large quantity of troublesome workloads is reduced, and the efficiency is increased.
Description
Technical field
The present invention relates to directed embedding techniques.
Background technology
Because fish yolk enriches when the section of conventional light microscopic can not cutting, another receives the limitation of light microscope and Electronic Speculum and the influence of slice thickness, when selecting fish fertilization part, will need part (being often referred to the part of being fertilized) to lose easily.Light microscopic of directly processing or Electronic Speculum sample because the section area is little, are repaiied multiple reasons such as piece is inaccurate, and the part that will need is easily again repaiied and lost, and causes imperfect phenomenon, directly influences observation, must do great amount of samples like this and just can observe needed part.
Summary of the invention
The invention provides the directed embedding techniques of fish oosperm, purpose is that the fertilization part is prone to lose and the ropy problem of cutting in the existing film-making in order to solve.
The directed embedding techniques of fish oosperm carries out according to the following steps: one, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut, and ovum is processed the 2mm fritter, adopts the directed embedding of secondary to process the light microscopic embedded block of routine then;
Four, the light microscopic embedded block adopts conventional microtomy to cut into slices, and processes conventional light microscopic slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
The directed embedding techniques of fish oosperm carries out according to the following steps: one, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut; Ovum is made the 1mm fritter; After Tuo Latuo benzene is handled, adopting osmic acid to carry out secondary fixes; Wash with phosphate buffer then; Carry out processed with each grade acetone again; Carry out directed embedding and polymerization with epoxy resin #618 then, obtain the Electronic Speculum embedded block;
Four, the Electronic Speculum embedded block adopts the electron microscopic section technology to cut into slices, and processes conventional Electronic Speculum slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
The directed embedding techniques of fish oosperm of the present invention; The light microscopic of processing or the sample of Electronic Speculum can guarantee that fish-egg is can cutting complete, and the fertilization part of fish-egg can be observed needed content accurately; The cutting quality is good; Clear picture, observing effect is good, makes the microcosmic and the structure of macroscopic view that a unified understanding arranged; Reduced the workload of a large amount of tired ropes simultaneously, raised the efficiency.
Description of drawings
Fig. 1 is the micrograph of the crucian embryonated egg light microscopic slice, thin piece that gained is complete among the embodiment 1; Fig. 2 is the micrograph of gained crucian embryonated egg animal pole light microscopic slice, thin piece among the embodiment 1; Fig. 3 is the micrograph of gained crucian embryonated egg polar body Electronic Speculum slice, thin piece among the embodiment 2; Fig. 4 is the micrograph of gained crucian embryonated egg both sexes nuclear power eyeglass among the embodiment 2.
Embodiment
Embodiment one: the directed embedding techniques of this embodiment fish oosperm carries out according to the following steps:
One, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut, and ovum is processed the 2mm fritter, adopts the directed embedding of secondary to process the light microscopic embedded block of routine then;
Four, the light microscopic embedded block adopts conventional microtomy to cut into slices, and processes conventional light microscopic slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
Fish-egg is the fish-egg of all fish in this embodiment step 1.
Processed is that 70% ethanol, volumetric concentration are that 85% ethanol, volumetric concentration are that 75% ethanol and volumetric concentration are that 100% ethanol is handled for adopting volumetric concentration successively in this embodiment step 1.
Transparent processing is 100% ethanol for the employing volumetric concentration in this embodiment step 1: xylene=handle at 1: 1, handle with xylene then.
Pass through wax in this embodiment step 1 and be treated to employing xylene: paraffin=handle at 1: 1, handle with paraffin then.
Reflective localization method in this embodiment step 2; Be embedded block to be fixed be put under the stereo microscope; Regulate light source then, make between tangent plane and the surface level of ovum of embedded block to become 28～32 degree, it is reflective that tangent plane is occurred; Observe its structure and choose the fertilization part, position with sharp cutter then and repair piece.
Hot blade is meant the blade that burns heat with alcolhol burner in the present embodiment step 3.
Embodiment two: the directed embedding techniques of this embodiment fish oosperm carries out according to the following steps: one, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut; Ovum is made the 1mm fritter; After Tuo Latuo benzene is handled, adopting osmic acid to carry out secondary fixes; Wash with phosphate buffer then; Carry out processed with each grade acetone again; Carry out directed embedding and polymerization with epoxy resin #618 then, obtain the Electronic Speculum embedded block;
Four, the Electronic Speculum embedded block adopts the electron microscopic section technology to cut into slices, and processes conventional Electronic Speculum slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
Fish-egg is the fish-egg of all fish in this embodiment step 1.
Processed is that 70% ethanol, volumetric concentration are that 85% ethanol, volumetric concentration are that 75% ethanol and volumetric concentration are that 100% ethanol is handled for adopting volumetric concentration successively in this embodiment step 1.
Transparent processing is 100% ethanol for the employing volumetric concentration in this embodiment step 1: xylene=handle at 1: 1, handle with xylene then.
Pass through wax in this embodiment step 1 and be treated to employing xylene: paraffin=handle at 1: 1, handle with paraffin then.
Reflective localization method in this embodiment step 2; Be embedded block to be fixed be put under the stereo microscope; Regulate light source then, make between tangent plane and the surface level of ovum of embedded block to become 28～32 degree, it is reflective that tangent plane is occurred; Observe its structure and choose the fertilization part, position with sharp cutter then and repair piece.
Hot blade is meant the blade that burns heat with alcolhol burner in the present embodiment step 3.
The compound method of osmic acid: 4%K in this embodiment step 3
2CrO
7Potassium dichromate (PH7.2) 5ml+2%O
sO
4Osmium tetroxide WS 10ml+3.4%NaCl sodium-chloride water solution 5ml mix immobile liquid, make to contain 1% potassium dichromate, 1% osmium tetroxide and 0.85% sodium chloride in the immobile liquid final PH=7.2.
The compound method of phosphate buffer in this embodiment step 3: the Na that 1. gets 11.876g
2HPO
4Surely be dissolved in the 1000ml aquae destillata, obtain solution A; 2. get the KH of 4.531g
2PO
4Potassium dihydrogen phosphate is dissolved in the 500ml aquae destillata surely, obtains solution B; 3. solution A and solution B mixing are promptly got.
To carry out processed be that to adopt volumetric concentration successively be that 50% acetone, volumetric concentration are that 70% acetone, volumetric concentration are that 95% acetone and volumetric concentration are that 100% acetone carries out processed to each grade acetone in this embodiment step 3.
Embodiment 1
The directed embedding techniques of fish oosperm carries out according to the following steps:
One, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut, and ovum is processed the 2mm fritter, adopts the directed embedding of secondary to process the light microscopic embedded block of routine then;
Four, the light microscopic embedded block adopts conventional microtomy to cut into slices, and processes conventional light microscopic slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
Fish-egg adopts the fish-egg of crucian in the present embodiment step 1.
Gained light microscopic slice, thin piece is as illustrated in fig. 1 and 2 through om observation in the present embodiment, fish-egg can cutting complete and clear picture, sharp outline, the fertilization part of fish-egg can be observed needed content accurately.
Embodiment 2
The directed embedding techniques of fish oosperm carries out according to the following steps:
One, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut; Ovum is made the 1mm fritter; After Tuo Latuo benzene is handled, adopting osmic acid to carry out secondary fixes; Wash with phosphate buffer then; Carry out processed with each grade acetone again; Carry out directed embedding and polymerization with epoxy resin #618 then, obtain the Electronic Speculum embedded block;
Four, the Electronic Speculum embedded block adopts the electron microscopic section technology to cut into slices, and processes conventional Electronic Speculum slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
Fish-egg adopts the fish-egg of crucian in the present embodiment step 1.
Gained Electronic Speculum slice, thin piece in the present embodiment, through electron microscopic observation, shown in Fig. 3 and 4, fish-egg can cutting complete and clear picture, sharp outline, the fertilization part of fish-egg can be observed needed content accurately.
Claims (2)
1. the directed embedding techniques of fish oosperm; It is characterized in that the directed embedding techniques of fish oosperm carries out according to the following steps: one, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut, and ovum is processed the 2mm fritter, adopts the directed embedding of secondary to process the light microscopic embedded block of routine then;
Four, the light microscopic embedded block adopts conventional microtomy to cut into slices, and processes conventional light microscopic slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
2. the directed embedding techniques of fish oosperm; It is characterized in that the directed embedding techniques of fish oosperm carries out according to the following steps: one, fish-egg being put into mass concentration is that 10% formaldehyde is fixing, carries out processed, the transparent processing before the embedding then successively and passes through wax and handle;
Two, fish-egg carries out embedding after step 1 is handled, and embedded block is complied with reflective localization method and positioned and repair piece through slightly repairing piece after;
Three, the fish-egg of repairing piece through the location is with hot blade cut mark fertilization part and cut; Ovum is made the 1mm fritter; After Tuo Latuo benzene is handled, adopting osmic acid to carry out secondary fixes; Wash with phosphate buffer then; Carry out processed with each grade acetone again; Carry out directed embedding and polymerization with epoxy resin #618 then, obtain the Electronic Speculum embedded block;
Four, the Electronic Speculum embedded block adopts the electron microscopic section technology to cut into slices, and processes conventional Electronic Speculum slice, thin piece, promptly accomplishes the directed embedding of fish oosperm.
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| CN2012100651130A CN102607930A (en) | 2012-03-13 | 2012-03-13 | Fish germ cell orientated embedding technology |
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| CN2012100651130A CN102607930A (en) | 2012-03-13 | 2012-03-13 | Fish germ cell orientated embedding technology |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558055A (en) * | 2013-09-29 | 2014-02-05 | 中国疾病预防控制中心病毒病预防控制所 | Positioning ultrathin slice method for pathologic cell infected with virus |
| CN105571916A (en) * | 2015-12-24 | 2016-05-11 | 四川省农业科学院水产研究所 | Acipenser dabryanus oosperm tissue slicing method |
| CN110082380A (en) * | 2019-05-13 | 2019-08-02 | 曾琪琪 | A method of it is connected with cell between In Situ Tem Study cell monolayer |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558055A (en) * | 2013-09-29 | 2014-02-05 | 中国疾病预防控制中心病毒病预防控制所 | Positioning ultrathin slice method for pathologic cell infected with virus |
| CN103558055B (en) * | 2013-09-29 | 2016-01-06 | 中国疾病预防控制中心病毒病预防控制所 | A kind of locating super-thin dicing method for infecting viral sick cell |
| CN105571916A (en) * | 2015-12-24 | 2016-05-11 | 四川省农业科学院水产研究所 | Acipenser dabryanus oosperm tissue slicing method |
| CN105571916B (en) * | 2015-12-24 | 2018-08-17 | 四川省农业科学院水产研究所 | A kind of tissue section method of acipenser dabryanus fertilized eggs |
| CN110082380A (en) * | 2019-05-13 | 2019-08-02 | 曾琪琪 | A method of it is connected with cell between In Situ Tem Study cell monolayer |
| CN110082380B (en) * | 2019-05-13 | 2022-04-08 | 曾琪琪 | Method for in-situ observation of intercellular cell junction of single layer by transmission electron microscope |
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Application publication date: 20120725 |