CN106404475A - A method of preparing a dactylis glomerata root tissue paraffin section - Google Patents
A method of preparing a dactylis glomerata root tissue paraffin section Download PDFInfo
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Abstract
A method of preparing a dactylis glomerata root tissue paraffin section is disclosed. The method includes a step of killing and immobilizing a fresh dactylis glomerata root tissue block, a step of washing, a step of dehydrating and hyalinizing, a step of wax soaking and embedding, a step of block trimming and sectioning, a step of section expanding and roasting, a step of dewaxing, rehydrating and staining with safranine, a step of staining with fast green, dehydrating and hyalinizing and a step of section sealing. After the step of washing and before the step of dehydrating and hyalinizing, agarose pre-embedding is performed and includes embedding the washed dactylis glomerata root tissue block into agarose gel having a content of 5% by mass and melt by stewing. The dactylis glomerata root tissue paraffin section prepared by the method is complete in tissue, clear in cell boundary, bright in color, sharp in color contrast and clean in sectioning.
Description
Technical field
The present invention relates to paraffin section technical field is and in particular to the method for orchardgrass root tissue paraffin section.
Background technology
Paraffin method is using paraffin as embedding medium, through a series of methods processing and making Permanent slide.Material not only can be cut into very thin section by it（Less than 4 μm）, and serial section can be made, and persistence, definition is good, is also suitable for making cytology research.Paraffin section is most important a kind of the most frequently used method, extensive application in the production and scientific research of phytotomy and its association area on microscopic examination technique.
Especially in recent years, with the rise of scientific and technical development and ecological plant anatomy, paraffin section becomes different niches plant or the important means of anthropogenic sere sociales ecological Adaptability.But, it is long to there is the film-making cycle in traditional paraffin section method, easily subject to conditions, more using chemical reagent in film-making, have certain murder by poisoning to people, so that material is become fragile, hardening or deformation, some inclusions are easy to disappear, so that tissue is diminished, or even make tissue torsional deflection, cell contained substance reduces and refractivity strengthens, being accurate in one's observation property of impact, and film-making cost higher the problems such as, if adding, the piece of tissue being taken is less, how fast and accurately to embed be also successfully film-making key in place of.
Content of the invention
Present invention aim at providing a kind of preparation method of orchardgrass root tissue paraffin section.The orchardgrass root tissue paraffin section tissue that the method is obtained is complete, cell boundaries are clear.
The object of the invention is achieved through the following technical solutions：
A kind of preparation method of orchardgrass root tissue paraffin section, including to fresh duck Rhizoma Imperatae portion piece of tissue killing with fixing, rinse, dehydration with transparent, waxdip and embedding, repair block and section, exhibition piece and roasting piece, dewaxing, rehydration and sarranine dyeing, fast green dyeing, dehydration and transparent, and mounting step；It is characterized in that：After rinsing step, dehydration with transparent step before, also carry out agarose pre-embedding, described agarose pre-embedding be by rinse after orchardgrass root tissue block be embedded in through in 5% agarose gel after enduring, in terms of weight/mass percentage composition（5 g agaroses are dissolved in 100 mL distilled water）.Solve the problems, such as to be difficult to quick and precisely embed because orchardgrass root tissue is too small it was found that, carrying out described agarose pre-embedding and processing.
Optimize further, after killing and be fixing, before flushing, be also acidified, specifically the orchardgrass fixing root tissue block is placed in 0.5 mol/L solution of trichloroacetic acid, is acidified 6-7 d, in acidization, during 3-4 d, change a 0.5 mol/L solution of trichloroacetic acid.
Further, above-mentioned kill with fixing step in, the fixative of employing is FAA mixed stationary liquid, as formalin：Glacial acetic acid：70 % ethanol（Volumetric concentration）=1:1:18th, with volume basis.
Preferably, dehydration and transparent step are that the orchardgrass root tissue block after above-mentioned agarose pre-embedding is sequentially placed into volume ratio for 1:5:4 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 2:5:3 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 3:5:2 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 5:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in volume ratio 15:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in 2 h in the pure butanol of analysis, then are placed in 2 h in the pure butanol of new analysis.
Further, in above-mentioned waxdip and embedding step, the volume ratio that the orchardgrass root tissue block after dehydration and transparent processing is put in 42 DEG C is 1:In 1 dimethylbenzene and soft wax intermixture, point I, II two-stage, each 1 hour；It is then transferred to waxdip 6 hours in 53 DEG C of pure soft waxs, point I, II two-stage, each 3 hours；Transfer to waxdip 3 hours in 63 DEG C of pure hard waxes, point I, II two-stage, each 1.5 hours；Described soft wax is the paraffin of 46-48 DEG C of fusing point, and described hard wax is 56-58 DEG C of paraffin for fusing point.Above-mentioned I, II two-stage refers to be repeated twice, and changes every time and new does not have used corresponding wax liquor.
Further, in above-mentioned exhibition piece and roasting piece step, for opening up, albumen glycerol alite paste is being coated on the microscope slide of piece, described albumen glycerol alite paste is 1 by volume ratio:1 Ovum Gallus domesticus album and glycerol composition.
Further, in above-mentioned dewaxing, rehydration and sarranine staining procedure, dewaxed three times using dimethylbenzene, 25 minutes every time；Described rehydration sequentially enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, 95% ethanol, 85% ethanol, in 70% ethanol, ethanol be concentration expressed in percentage by volume, often step stops 4-5 minute, then distillation washing entered 1% aqueous solution sarranine dye liquor, mass concentration after 5 seconds, and sealing is overnight.Above-mentioned I, II, III grade refers in triplicate, changes every time and new does not have used dehydrated alcohol.
Further, in above-mentioned fast green dyeing, dehydration and transparent step, will float the removal floating color 5-10 second in 95% ethanol through the section that dewaxing, rehydration and sarranine dyeing are processed, the fast green dye liquor of 95% ethanol being 0.5% subsequently into mass concentration, keep 30 seconds, described 95% ethanol is concentration expressed in percentage by volume；Then it is dehydrated：Through three-level pure analysis absolute ethyl alcohol process, as enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, every grade 30 seconds -1 minute；Transparent processing is entered after dehydration：Process through the three-level pure dimethylbenzene of analysis, as enter I grade of dimethylbenzene, II grade of dimethylbenzene, III grade of dimethylbenzene, every grade 20 minutes.Above-mentioned I, II, III grade refers in triplicate, changes every time and new does not have used dehydrated alcohol or dimethylbenzene.
Most preferably say, a kind of preparation method of orchardgrass root tissue paraffin section, as follows：
（1）Kill and fix
Clip fresh duck Rhizoma Imperatae portion, cuts orchardgrass root tissue block with disposable double-edged razor blade, about 2 ~ 3 mm, after to be gently placed in FAA mixed stationary liquid fixing, described FAA mixed stationary liquid immediately be volume ratio is 1:1:18 formalin, glacial acetic acid, 70 % ethanol；Fixative volume should be 10 ~ 15 times of orchardgrass root tissue block cumulative volume.Material after fixation, if standby using needing to preserve not in time；Orchardgrass root tissue and is placed in 4 DEG C of refrigerator and can preserve half a year to 1 year in above-mentioned FAA mixed stationary liquid, and the structure of material will not change and also be unlikely to degenerate.Described killing effectively prevent cell tissue generation autolysis with fixation.
（2）Acidifying
The orchardgrass fixing root tissue block is placed in 0.5 mol/L solution of trichloroacetic acid, is acidified 6-7 d, in acidization, during 3-4 d, change a 0.5 mol/L solution of trichloroacetic acid.
（3）Rinse
The clean double gauze of clip is converted into square, using tiny tweezers, acidified orchardgrass root tissue block is placed in gauze central authorities, is carried out after title material and numbering labelling with 2 B pencils, gauze is closed up, with nylon rope, gauze is tied up, do not spilt in the water of flowing by material and be defined；Little for gauze group is placed in the water of flowing, rinses about 24 h.
（4）Agarose pre-embedding
Prepare the agarose gel that mass percentage concentration is 5 %（5 g agaroses are taken to be dissolved in 100 mL distilled water）, after repeatedly enduring, pour on the flat board of prepositioned 4 hole card punch, thickness about 5 ~ 7 mm, in case material is sealed after putting into；After agarose condensation, take out card punch, take out crumpled gauze from the water of flowing and open crumpled gauze, by the orchardgrass root tissue block cross section after the completion of rinsing upwards, put down gently to gel pore bottom with tiny tweezers successively（Hole will not be punched by card punch, and orchardgrass root tissue block will not spill from gel pore bottom）, and agarose is trimmed to regular strip, cut the upper left corner to show material number, then the sealing in agar hole is carried out on alcohol burner, avoids sealing large quantity of air in the hole.
（5）Dehydration with transparent
Gently gripping the orchardgrass root tissue agar block embedding with tweezers and being sequentially placed into volume ratio is 1:5:4 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 2:5:3 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 3:5:2 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 5:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in volume ratio 15:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in 2 h in the pure butanol of analysis, then are placed in 2 h in the pure butanol of new analysis.
（6）Waxdip and embedding
Pour dimethylbenzene in the beaker dried, then pouring the liquid soft wax of equivalent into, volume ratio is obtained is 1：1 dimethylbenzene, soft wax intermixture, are placed in standby in 42 DEG C of calorstats；Grip transparent completely orchardgrass root tissue Agarose plug with thin tweezers and put down dimethylbenzene in 42 DEG C of calorstats, soft wax intermixture gently, point I, II two-stage, each 1 h；Then Agarose plug is transferred to pure soft wax waxdip 6 h in 53 DEG C of calorstats, point I, II two-stage, each 3 h；Transfer to pure hard wax waxdip 3 h in 63 DEG C of calorstats, point I, II two-stage, each 1.5 h.Above-mentioned I, II two-stage refers to be repeated twice, and changes every time and new does not have used corresponding wax liquor.
In the embedding carton got ready, pour the hard wax in pure hard wax II into, the Agarose plug that gripping waxdip completes simultaneously, ajust tangent plane, hard wax surface in carton to be embedded starts to be condensed into solid, then immediately whole embedding carton is placed in cold water so as to be frozen into wax stone at once.
Above-mentioned paraffin smooth uniform and free from admixture, are provided by Shanghai Yong Hua paraffin company limited.Above-mentioned soft wax is the paraffin of 46 ~ 48 DEG C of fusing point, and hard wax is the paraffin of 56 ~ 58 DEG C of fusing point.
（7）Repair block and section
Cut off embedding carton, paraffin mass is taken out, with repairing block knife, paraffin mass is accomplished terrace edge shape, upper and lower tangent plane is parallel, avoids piece of tissue, prevent injury tissue block, be then attached on hard scantling block；With microtome clamping wooden unit, make wax stone parallel with the edge of a knife, then cut into slices, slice thickness is advisable with 5 ~ 7 μm.
（8）Exhibition piece and roasting piece
Through above-mentioned repair block, the wax stone after section becomes the wax band of a continuous strip, choose complete wax band and cut into segment, first smearing last layer on clean microscope slide by volume ratio is 1:The albumen glycerol alite paste of 1 Ovum Gallus domesticus album and glycerol composition, effectively prevent from opening up piece process wax band and come off, more lightly wax band is positioned in the large beaker filling distilled water with tiny tweezers（It is pre-placed in 49 ~ 50 DEG C of thermostat water baths, keep water temperature in large beaker consistent with thermostat water bath water temperature）Extend piece, carry out dragging for piece with the microscope slide being covered with described albumen glycerol, ajust wax band position with pincet（At 1/3 ~ 2/3 under microscope slide）, suck unnecessary moisture with absorbent paper, and carry out labelling with Pencil with 2B hardness；Then the microscope slide being covered with wax band is placed in slide holding frame, in 37 DEG C of calorstats, dries piece 24 h, or natural air drying.
（9）Dewaxing, rehydration and sarranine dyeing
Above-mentioned dried section is dewaxed three times with dimethylbenzene, 25 minutes every time；After dewaxing finishes, that is, enter rehydration and sarranine dyeing series：Sequentially enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, 95% ethanol, 85% ethanol, in 70% ethanol, ethanol be concentration expressed in percentage by volume, often step stops 4-5 minute, then distillation washing entered 1% aqueous solution sarranine dye liquor, mass concentration after 5 seconds, and sealing is overnight.Above-mentioned I, II, III grade refers in triplicate, changes every time and new does not have used dehydrated alcohol.
（10）Fast green dyeing, dehydration with transparent
To float the removal floating color 5-10 second in 95% ethanol through the section that dewaxing, rehydration and sarranine dyeing are processed, the fast green dye liquor of 95% ethanol being 0.5% subsequently into mass concentration, keep 30 seconds, described 95% ethanol is concentration expressed in percentage by volume；Then it is dehydrated：Through three-level pure analysis absolute ethyl alcohol process, as enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, every grade 30 seconds -1 minute；Transparent processing is entered after dehydration：Process through the three-level pure dimethylbenzene of analysis, as enter I grade of dimethylbenzene, II grade of dimethylbenzene, III grade of dimethylbenzene, every grade 20 minutes.Above-mentioned I, II, III grade refers in triplicate, changes every time and new does not have used dehydrated alcohol or dimethylbenzene.
（11）Mounting
Above-mentioned transparent good section is taken out from dimethylbenzene, wipes clean unnecessary dimethylbenzene with gauze, then use neutral gum mounting, near piece of tissue to be observed, drip mountant, along lightly covered（Prevent bubble）, can be in Olympus basis of microscopic observation and shooting after last natural air drying.
The present invention has the beneficial effect that：
The orchardgrass root tissue paraffin section that the inventive method is obtained, tissue is complete, cell boundary is clear, bright in luster, color contrast is distinct, section is clean for it, show that this method is applied to orchardgrass root tissue paraffin section and makes, can have more preferable effect than traditional plant paraffin section manufacture method.And the making of relatively high-volume orchardgrass root tissue paraffin section can be completed within a short period of time, test effect is good.
Brief description
Fig. 1：' the northern regions of the Yunnan Province ' orchardgrass lateral root tissue slice figure；
Fig. 2：' PI 594994 ' orchardgrass primary root tissue slice figure；
Fig. 3：' PG 49 ' orchardgrass primary root tissue slice figure；
Fig. 4：' Guizhou Province grass 4 ' orchardgrass primary root tissue slice figure.
Specific embodiment
Below by specific embodiment, the present invention is specifically described; it is pointed out here that following examples are served only for the present invention is further described; it is not intended that limiting the scope of the invention, the person skilled in the art of this area can make some according to foregoing invention content and nonessential improve and adjust to the present invention.
' the northern regions of the Yunnan Province ', ' PI 594994 ', ' PG 49 ' adopting in following examples, ' Guizhou Province grass 4 ' are orchardgrass kind（System）Title, is respectively derived from Sichuan Agricultural University, U.S.'s forage germplasm resources storehouse, New Zealand, Guizhou Province Grass Industry Research Institute；Kind well known in the art（System）.
Embodiment 1
A kind of preparation method of orchardgrass root tissue paraffin section, as follows：
（1）Kill and fix
Clip fresh ' the northern regions of the Yunnan Province ' orchardgrass lateral root portion, cuts orchardgrass root tissue block with disposable double-edged razor blade, about 2 ~ 3 mm, after to be gently placed in FAA mixed stationary liquid fixing, described FAA mixed stationary liquid immediately be volume ratio is 1:1:18 formalin, glacial acetic acid, 70 % ethanol；Fixative volume should be 10 ~ 15 times of orchardgrass root tissue block cumulative volume.Material after fixation, if standby using needing to preserve not in time；Orchardgrass root tissue and is placed in 4 DEG C of refrigerator and can preserve half a year to 1 year in above-mentioned FAA mixed stationary liquid, and the structure of material will not change and also be unlikely to degenerate.Described killing effectively prevent cell tissue generation autolysis with fixation.
（2）Acidifying
The orchardgrass fixing root tissue block is placed in 0.5 mol/L solution of trichloroacetic acid, is acidified 6-7 d, in acidization, during 3-4 d, change a 0.5 mol/L solution of trichloroacetic acid.
（3）Rinse
The clean double gauze of clip is converted into square, using tiny tweezers, acidified orchardgrass root tissue block is placed in gauze central authorities, is carried out after title material and numbering labelling with 2 B pencils, gauze is closed up, with nylon rope, gauze is tied up, do not spilt in the water of flowing by material and be defined；Little for gauze group is placed in the water of flowing, rinses about 24 h.
（4）Agarose pre-embedding
Prepare the agarose gel that mass percentage concentration is 5 %（5 g agaroses are taken to be dissolved in 100 mL distilled water）, after repeatedly enduring, pour on the flat board of prepositioned 4 hole card punch, thickness about 5 ~ 7 mm, in case material is sealed after putting into；After agarose condensation, take out card punch, take out crumpled gauze from the water of flowing and open crumpled gauze, by the orchardgrass root tissue block cross section after the completion of rinsing upwards, put down gently to gel pore bottom with tiny tweezers successively（Hole will not be punched by card punch, and orchardgrass root tissue block will not spill from gel pore bottom）, and agarose is trimmed to regular strip, cut the upper left corner to show material number, then the sealing in agar hole is carried out on alcohol burner, avoids sealing large quantity of air in the hole.
（5）Dehydration with transparent
Gently gripping the orchardgrass root tissue agar block embedding with tweezers and being sequentially placed into volume ratio is 1:5:4 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 2:5:3 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 3:5:2 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 5:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in volume ratio 15:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in 2 h in the pure butanol of analysis, then are placed in 2 h in the pure butanol of new analysis.
（6）Waxdip and embedding
Pour dimethylbenzene in the beaker dried, then pouring the liquid soft wax of equivalent into, volume ratio is obtained is 1：1 dimethylbenzene, soft wax intermixture, are placed in standby in 42 DEG C of calorstats；Grip transparent completely orchardgrass root tissue Agarose plug with thin tweezers and put down dimethylbenzene in 42 DEG C of calorstats, soft wax intermixture gently, point I, II two-stage, each 1 h；Then Agarose plug is transferred to pure soft wax waxdip 6 h in 53 DEG C of calorstats, point I, II two-stage, each 3 h；Transfer to pure hard wax waxdip 3 h in 63 DEG C of calorstats, point I, II two-stage, each 1.5 h.Above-mentioned I, II two-stage refers to be repeated twice, and changes every time and new does not have used corresponding wax liquor.
In the embedding carton got ready, pour the hard wax in pure hard wax II into, the Agarose plug that gripping waxdip completes simultaneously, ajust tangent plane, hard wax surface in carton to be embedded starts to be condensed into solid, then immediately whole embedding carton is placed in cold water so as to be frozen into wax stone at once.
Above-mentioned paraffin smooth uniform and free from admixture, are provided by Shanghai Yong Hua paraffin company limited.Above-mentioned soft wax is the paraffin of 46 ~ 48 DEG C of fusing point, and hard wax is the paraffin of 56 ~ 58 DEG C of fusing point.
（7）Repair block and section
Cut off embedding carton, paraffin mass is taken out, with repairing block knife, paraffin mass is accomplished terrace edge shape, upper and lower tangent plane is parallel, avoids piece of tissue, prevent injury tissue block, be then attached on hard scantling block；With microtome clamping wooden unit, make wax stone parallel with the edge of a knife, then cut into slices, slice thickness is advisable with 5 ~ 7 μm.
（8）Exhibition piece and roasting piece
Through above-mentioned repair block, the wax stone after section becomes the wax band of a continuous strip, choose complete wax band and cut into segment, first smearing last layer on clean microscope slide by volume ratio is 1:The albumen glycerol alite paste of 1 Ovum Gallus domesticus album and glycerol composition, effectively prevent from opening up piece process wax band and come off, more lightly wax band is positioned in the large beaker filling distilled water with tiny tweezers（It is pre-placed in 49 ~ 50 DEG C of thermostat water baths, keep water temperature in large beaker consistent with thermostat water bath water temperature）Extend piece, carry out dragging for piece with the microscope slide being covered with described albumen glycerol, ajust wax band position with pincet（At 1/3 ~ 2/3 under microscope slide）, suck unnecessary moisture with absorbent paper, and carry out labelling with Pencil with 2B hardness；Then the microscope slide being covered with wax band is placed in slide holding frame, in 37 DEG C of calorstats, dries piece 24 h, or natural air drying.
（9）Dewaxing, rehydration and sarranine dyeing
Above-mentioned dried section is dewaxed three times with dimethylbenzene, 25 minutes every time；After dewaxing finishes, that is, enter rehydration and sarranine dyeing series：Sequentially enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, 95% ethanol, 85% ethanol, in 70% ethanol, ethanol be concentration expressed in percentage by volume, often step stops 4-5 minute, then distillation washing entered 1% aqueous solution sarranine dye liquor, mass concentration after 5 seconds, and sealing is overnight.Above-mentioned I, II, III grade refers in triplicate, changes every time and new does not have used dehydrated alcohol.
（10）Fast green dyeing, dehydration with transparent
To float the removal floating color 5-10 second in 95% ethanol through the section that dewaxing, rehydration and sarranine dyeing are processed, the fast green dye liquor of 95% ethanol being 0.5% subsequently into mass concentration, keep 30 seconds, described 95% ethanol is concentration expressed in percentage by volume；Then it is dehydrated：Through three-level pure analysis absolute ethyl alcohol process, as enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, every grade 30 seconds -1 minute；Transparent processing is entered after dehydration：Process through the three-level pure dimethylbenzene of analysis, as enter I grade of dimethylbenzene, II grade of dimethylbenzene, III grade of dimethylbenzene, every grade 20 minutes.Above-mentioned I, II, III grade refers in triplicate, changes every time and new does not have used dehydrated alcohol or dimethylbenzene.
（11）Mounting
Above-mentioned transparent good section is taken out from dimethylbenzene, wipes clean unnecessary dimethylbenzene with gauze, then use neutral gum mounting, near piece of tissue to be observed, drip mountant, along lightly covered（Prevent bubble）, i.e. in Olympus basis of microscopic observation and shooting after last natural air drying, see Fig. 1.
Embodiment 2-4
It is respectively adopted ' PI 594994 ' orchardgrass primary root, ' PG 49 ' orchardgrass primary root, ' Guizhou Province grass 4 ' orchardgrass primary root carries out film-making.Other are with embodiment 1 method with result is as in Figure 2-4.Locate as see arrows 17 in fig 3：There is keratinization in older primary root or visible crust confluent monolayer cells, assume " fish scale-shaped " of bright green.Locate cell wall lignifying and the bolt matter of visible crust confluent monolayer cells shown in Fig. 4 arrow, and its endothelium confluent monolayer cells is in " shape of a hoof ".Its tissue is complete, section is clean, cell boundary is clear, bright in luster, color contrast is distinct.
Claims (9)
1. a kind of preparation method of orchardgrass root tissue paraffin section, including to fresh duck Rhizoma Imperatae portion piece of tissue killing with fixing, rinse, dehydration with transparent, waxdip and embedding, repair block and section, exhibition piece and roasting piece, dewaxing, rehydration and sarranine dyeing, fast green dyeing, dehydration and transparent, and mounting step；It is characterized in that：After rinsing step, dehydration with transparent step before, also carry out agarose pre-embedding, described agarose pre-embedding be by rinse after orchardgrass root tissue block be embedded in through in 5% agarose gel after enduring, in terms of weight/mass percentage composition.
2. preparation method as claimed in claim 1 it is characterised in that：After killing and be fixing, before flushing, also it is acidified, specifically the orchardgrass fixing root tissue block is placed in 0.5 mol/L solution of trichloroacetic acid, be acidified 6-7 d, in acidization, during 3-4 d, changed a 0.5 mol/L solution of trichloroacetic acid.
3. preparation method as claimed in claim 1 or 2 it is characterised in that：Described kill with fixing step in, the fixative of employing is FAA mixed stationary liquid, as formalin：Glacial acetic acid：70 % ethanol（Volumetric concentration）=1:1:18th, with volume basis.
4. preparation method as claimed in claim 2 it is characterised in that：Dehydration and transparent step are that the orchardgrass root tissue block after described agarose pre-embedding is sequentially placed into volume ratio for 1:5:4 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 2:5:3 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 3:5:2 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 5:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in volume ratio 15:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in 2 h in the pure butanol of analysis, then are placed in 2 h in the pure butanol of analysis.
5. preparation method as claimed in claim 3 it is characterised in that：In described waxdip and embedding step, the volume ratio that the orchardgrass root tissue block after dehydration and transparent processing is put in 42 DEG C is 1:In 1 dimethylbenzene and soft wax intermixture, point I, II two-stage, each 1 hour；It is then transferred to waxdip 6 hours in 53 DEG C of pure soft waxs, point I, II two-stage, each 3 hours；Transfer to waxdip 3 hours in 63 DEG C of pure hard waxes, point I, II two-stage, each 1.5 hours；Described soft wax is the paraffin of 46-48 DEG C of fusing point, and described hard wax is 56-58 DEG C of paraffin for fusing point.
6. preparation method as claimed in claim 4 it is characterised in that：In described exhibition piece and roasting piece step, for opening up, albumen glycerol alite paste is being coated on the microscope slide of piece, described albumen glycerol alite paste is 1 by volume ratio:1 Ovum Gallus domesticus album and glycerol composition.
7. preparation method as claimed in claim 5 it is characterised in that：In described dewaxing, rehydration and sarranine staining procedure, dewaxed three times using dimethylbenzene, 25 minutes every time；Described rehydration sequentially enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, 95% ethanol, 85% ethanol, in 70% ethanol, ethanol be concentration expressed in percentage by volume, often step stops 4-5 minute, then distillation washing entered 1% aqueous solution sarranine dye liquor, mass concentration after 5 seconds, and sealing is overnight.
8. preparation method as claimed in claim 7 it is characterised in that：In described fast green dyeing, dehydration and transparent step, to float the removal floating color 5-10 second in 95% ethanol through the section that dewaxing, rehydration and sarranine dyeing are processed, the fast green dye liquor of 95% ethanol being 0.5% subsequently into mass concentration, keeps 30 seconds, and described 95% ethanol is concentration expressed in percentage by volume；Then it is dehydrated：Through three-level pure analysis absolute ethyl alcohol process, as enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, every grade 30 seconds -1 minute；Transparent processing is entered after dehydration：Process through the three-level pure dimethylbenzene of analysis, as enter I grade of dimethylbenzene, II grade of dimethylbenzene, III grade of dimethylbenzene, every grade 20 minutes.
9. a kind of preparation method of orchardgrass root tissue paraffin section, as follows：
（1）Kill and fix
Clip fresh duck Rhizoma Imperatae portion, cuts orchardgrass root tissue block with disposable double-edged razor blade, about 2 ~ 3 mm, after to be immediately placed in FAA mixed stationary liquid fixing, described FAA mixed stationary liquid be volume ratio is 1:1:18 formalin, glacial acetic acid, 70 % ethanol；Fixative volume should be 10 ~ 15 times of orchardgrass root tissue block cumulative volume；
（2）Acidifying
The orchardgrass fixing root tissue block is placed in 0.5
In mol/L solution of trichloroacetic acid, it is acidified 6-7
D, in acidization, during 3-4 d, changes a 0.5 mol/L solution of trichloroacetic acid；
（3）Rinse
The clean double gauze of clip is converted into square, using tiny tweezers, acidified orchardgrass root tissue block is placed in gauze central authorities, is carried out after title material and numbering labelling with 2 B pencils, gauze is closed up, with nylon rope, gauze is tied up, do not spilt in the water of flowing by material and be defined；Little for gauze group is placed in the water of flowing, rinses 24 h；
（4）Agarose pre-embedding
Prepare the agarose gel that mass percentage concentration is 5 %, after repeatedly enduring, pour on the flat board of prepositioned 4 hole card punch, thickness about 5 ~ 7 mm, in case material is sealed after putting into；After agarose condensation, take out card punch, take out crumpled gauze and open crumpled gauze from the water of flowing, by the orchardgrass root tissue block cross section after the completion of rinsing upwards, put down gently to gel pore bottom with tiny tweezers successively, and agarose is trimmed to regular strip, cut the upper left corner to show material number, then the sealing in agar hole is carried out on alcohol burner；
（5）Dehydration with transparent
Gently gripping the orchardgrass root tissue agar block embedding with tweezers and being sequentially placed into volume ratio is 1:5:4 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 2:5:3 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 3:5:2 n-butyl alcohol, volumetric concentration are 95% ethanol, distill 2 h in water mixed liquid, and being placed in volume ratio is 5:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in volume ratio 15:4:1 n-butyl alcohol, volumetric concentration are 100% ethanol, distill 2 h in water mixed liquid, are placed in 2 h in the pure butanol of analysis, then are placed in 2 h in the pure butanol of new analysis；
（6）Waxdip and embedding
Pour dimethylbenzene in the beaker dried, then pouring the liquid soft wax of equivalent into, volume ratio is obtained is 1：1 dimethylbenzene, soft wax intermixture, are placed in standby in 42 DEG C of calorstats；Grip transparent completely orchardgrass root tissue Agarose plug with thin tweezers and put down dimethylbenzene in 42 DEG C of calorstats, soft wax intermixture gently, point I, II two-stage, each 1 h；Then Agarose plug is transferred to pure soft wax waxdip 6 h in 53 DEG C of calorstats, point I, II two-stage, each 3 h；Transfer to pure hard wax waxdip 3 h in 63 DEG C of calorstats, point I, II two-stage, each 1.5 h；
In the embedding carton got ready, pour the hard wax in pure hard wax II into, the Agarose plug that gripping waxdip completes simultaneously, ajust tangent plane, hard wax surface in carton to be embedded starts to be condensed into solid, then immediately whole embedding carton is placed in cold water so as to be frozen into wax stone at once；
（7）Repair block and section
Cut off embedding carton, paraffin mass is taken out, with repairing block knife, paraffin mass is accomplished terrace edge shape, upper and lower tangent plane is parallel, avoids piece of tissue, prevent injury tissue block, be then attached on hard scantling block；With microtome clamping wooden unit, make wax stone parallel with the edge of a knife, then cut into slices, 5 ~ 7 μm of slice thickness；
（8）Exhibition piece and roasting piece
Through described repair block, the wax stone after section becomes the wax band of a continuous strip, choose complete wax band and cut into segment, first smearing last layer on clean microscope slide by volume ratio is 1:1 Ovum Gallus domesticus album and the albumen glycerol alite paste of glycerol composition, lightly wax band is positioned in the large beaker fill distilled water with tiny tweezers again and extends piece, large beaker be positioned in 49 ~ 50 DEG C of thermostat water baths keep water temperature in large beaker consistent with thermostat water bath water temperature in advance, carry out dragging for piece with the microscope slide being covered with described albumen glycerol, ajust wax band position with pincet, suck unnecessary moisture with absorbent paper, and carry out labelling with Pencil with 2B hardness；Then the microscope slide being covered with wax band is placed in slide holding frame, in 37 DEG C of calorstats, dries piece 24 h, or natural air drying；
（9）Dewaxing, rehydration and sarranine dyeing
Described dried section is dewaxed three times with dimethylbenzene, 25 minutes every time；After dewaxing finishes, that is, enter rehydration and sarranine dyeing series：Sequentially enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, 95% ethanol, 85% ethanol, in 70% ethanol, ethanol be concentration expressed in percentage by volume, often step stops 4-5 minute, then distillation washing entered 1% aqueous solution sarranine dye liquor, mass concentration after 5 seconds, and sealing is overnight；
（10）Fast green dyeing, dehydration with transparent
To float the removal floating color 5-10 second in 95% ethanol through the section that dewaxing, rehydration and sarranine dyeing are processed, the fast green dye liquor of 95% ethanol being 0.5% subsequently into mass concentration, keep 30 seconds, described 95% ethanol is concentration expressed in percentage by volume；Then it is dehydrated：Through three-level pure analysis absolute ethyl alcohol process, as enter I grade of dehydrated alcohol, II grade of dehydrated alcohol, III grade of dehydrated alcohol, every grade 30 seconds -1 minute；Transparent processing is entered after dehydration：Process through the three-level pure dimethylbenzene of analysis, as enter I grade of dimethylbenzene, II grade of dimethylbenzene, III grade of dimethylbenzene, every grade 20 minutes；
（11）Mounting
Described transparent good section is taken out from dimethylbenzene, wipes clean unnecessary dimethylbenzene with gauze, then use neutral gum mounting, examine under a microscope after last natural air drying.
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| CN201610490667.3A CN106404475B (en) | 2016-06-29 | 2016-06-29 | A kind of preparation method of orchardgrass root tissue paraffin section |
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| CN109459262A (en) * | 2018-12-21 | 2019-03-12 | 东北农业大学 | A kind of preparation method that maturation maize root system is temporarily sliced |
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| CN109855936A (en) | 2019-06-07 |
| CN106404475B (en) | 2019-02-22 |
| CN109855936B (en) | 2020-08-11 |
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