CN102175482B - Method for slicing heavy leather blade paraffin - Google Patents
Method for slicing heavy leather blade paraffin Download PDFInfo
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- CN102175482B CN102175482B CN2011100010048A CN201110001004A CN102175482B CN 102175482 B CN102175482 B CN 102175482B CN 2011100010048 A CN2011100010048 A CN 2011100010048A CN 201110001004 A CN201110001004 A CN 201110001004A CN 102175482 B CN102175482 B CN 102175482B
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Abstract
The invention discloses a method for slicing heavy leather blade paraffin and relates to a method for slicing plant paraffin. Operating procedures include fixing, dehydrating, transparentizing, paraffin infiltration, embedding, slicing, dewaxing, dyeing and mounting. In the splicing process of the heavy leather blade paraffin, a fixing agent, a dehydrating procedure, a transparentizing procedure, a paraffin infiltration method, a dyeing method and mounting are improved, i.e., selection of the fixing agent, determination of the dehydrating procedure, determination of the transparentizing procedure, determination of the dewaxing procedure, the dyeing method and the mounting method are improved. The method is also suitable for other blades with different thicknesses and properties. By adopting the method, slice manufacturing of blade paraffin with different thicknesses and properties can be realized practically, and a uniform and practical manufacturing method for theoretical manufacturing of paraffin is popularized.
Description
Technical field
The present invention relates to a kind of plant paraffin section method, particularly a kind of heavy leather matter blade paraffin section method.
Background technology
Paraffin section is widely used in the conventional tabletting technology of histology, is mainly used in the morphosis of observation of cell tissue, but and long preservation.Paraffin section production process and link are various, and the cycle is long, and each link all can influence chipping qualities; For example occur easily in the microsection manufacture process problems such as section is crooked, section crackle (Wu Minhua, Ke Zhanhua. the FAQs of slicing processes and solution [J] Hainan Medical College journal in the paraffin wax flaking art, 2006,12 (3): 255); Though these problems can overcome; But, must make section again if material fragmentation but is to remedy.The reason of material fragmentation is traced back to many steps of paraffin manufacturing process; Comprise dehydration (Zhang Yuying. make problem and innovative approach [J] that paraffin section is prone to. Chinese science and technology information; 2007,3 (6): 91-92.), transparent, pass through wax (Chen Yewen. make the cognition [J] of paraffin organization section preparation. Xianning College's journal (medicine), 2006; 20 (3): 210-211.), embedding, section, dewaxing and mounting or the like; As long as one of them step makes mistakes, all possibly cause material fragmentation (king's kitchen range peace. plant microtechnic [M]. Beijing: agriculture publishing house, 1992:15-16.).Wherein dehydration, transparent, pass through steps such as wax, embedding, dewaxing and relate to factors such as reagent concentration, component ratio, soak time, processes such as section, dyeing and mounting relate to actual technical matters.Heavy leather matter blade is because blade is thick especially, and manufacturing process will be brought sizable difficulty.At first, reagent gets into organizes relatively difficulty, but can not achieve the goal with simple prolongation reagent soak time, because long soaking time can be brought adverse influence to material, such as the xylene long soaking time, can cause material fragility to increase, and organizes frangible.Secondly, dehydration also relatively is not easy, and dehydration thoroughly can not form empty wax disk(-sc) yet, material fragmentation occurs so heavy leather matter blade paraffin section is made regular meeting, causes the microsection manufacture failure.
Summary of the invention
The objective of the invention is to provides a kind of heavy leather matter blade paraffin section method to the problems referred to above that exist in the existing paraffin section method for making.
The present invention includes following steps:
1) with blade along master pulse two lateral incisions, with the nervate part of central authorities again crosscut become segment;
In step 1), said blade can adopt the mature leaf of fresh no disease and pest; Said with blade along master pulse two lateral incisions, available single-edge blade is the incision of each 5～6mm place along the master pulse both sides; The length of said segment can be 5～6mm.
2) the resulting blade of step 1) is fixed with fixing agent;
In step 2) in, said fixing agent can adopt omnipotent immobile liquid (also claim the FAA immobile liquid, prescription is 70% ethanol 90ml, glacial acetic acid 5ml, formalin 5ml); Said regular time is 24h at least.
3) with step 2) with the fixing blade dehydration of fixing agent;
In step 3); The concrete grammar of said dehydration can be: begin with 60% ethanol; 5% increases progressively; Every grade of 3～5h is specially 3～5h in 3～5h in 3～5h in 3～5h in 3～5h in 3～5h in 3～5h in 60% ethanol, 3～5h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol, 3～5h (for the first time) → 100% ethanol 3～5h (for the second time).
4) do transition with the solution of the different volumes ratio of xylene and ethanol; Every grade of 2～4h, detailed process is an xylene: 100% ethanol is in solution to soak 2～4h → xylene at 1: 2: 100% ethanol is in solution to soak 2～4h → xylene at 1: 1: 100% ethanol is in solution to soak in 2～4h → pure xylene soak at 2: 1 to soak 2～4h (for the second time) in 2～4h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly, and by volume, xylene: paraffin is 1: 1 solution transition waxdip 48～72h in 37～40 ℃ of constant temperature ovens; Change paraffin refined wax waxdip in 60～63 ℃ of constant temperature ovens afterwards into; Will change paraffin refined wax during this time 1 or 2 time, use template behind the wax thoroughly, 3 is a thickness; The big carton of folding 2cm * 2cm * 3cm is used for embedding;
6) section back exhibition sheet, paster is baked sheet then again;
In step 6), said section can be adopted the section of biologic slice machine, and the thickness of section can be 10～12 μ m; Said exhibition sheet can be earlier in 25%～35% ethanol transition, and the back is to open up sheet in the warm water in 48～55 ℃ in water temperature; Said paster can directly slightly dry by the fire sheet with ethanol lamp flame envelope; Said roasting sheet can be baked sheet 24～36h in 35～39 ℃ incubator.
7) dewaxing rehydration;
In step 7); The concrete grammar of said dewaxing rehydration is: by volume, 100% ethanol: xylene is 1～2min → 100% ethanol in 1: 2 solution: xylene is 1～2min → 100% ethanol in 1: 1 solution: xylene is 1～2min → 100% ethanol, 1～2min (for the first time) → 100% ethanol 1～2min (for the second time) → 95% ethanol, 1～2min → 85% ethanol, 1～2min → 70% ethanol, 1～2min → 50% ethanol, 1～2min in 2: 1 solution.
8) dyeing back mounting.
In step 8), the concrete grammar of said dyeing is: 1% sarranine solution-dyed, 2～4h → 95% ethanol, 1～2min → 100% ethanol, 1～2min (for the first time) → 100% ethanol (for the second time), 1～2min → 100% ethanol: xylene is 1～2min → 100% ethanol in 2: 1 solution: xylene is 1～2min → 100% ethanol in 1: 1 solution: xylene is 1～2min → xylene (for the first time), 10～15min → xylene (for the second time) 10～15min → 1% fast green 35～45s that redyes in 1: 2 solution; Said mounting can adopt canada balsam or transparent nail polish to carry out mounting.
Compare with the prior paraffin flaking method, the present invention has following outstanding advantage:
1) dehydration procedure adopts ethanol dehydration, begins with 60% ethanol, and 5% increases progressively, every grade of 3～5h.Concentration of alcohol at different levels 5% increase progressively, and increase the progression of ethanol, and thick coriaceous leaf is dewatered to thoroughly gradually.
2) confirming of transparent process: because heavy leather matter blade material is thicker; Xylene penetrates the comparison difficulty; Take different proportion (volume ratio) solution of xylene and ethanol to do transition; Specifically be followed successively by xylene: 100% ethanol is 1: 2 solution, xylene: 100% ethanol is 1: 1 solution, xylene: 100% ethanol is 2: 1 solution, pure xylene twice, every grade of 2～4h.
3) passing through wax, to adopt fusing point be 52～54 ℃ soft wax, and wax penetration process is used xylene: paraffin is 1: 1 mixed liquor transition waxdip 48～72h in 37～40 ℃ of constant temperature ovens, and this transition waxdip is the key of wax penetration process.
4) paster: do not use the bonding die agent, directly slightly dry by the fire sheet with ethanol lamp flame envelope.
5) dewaxing rehydration: take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 1～2min; Be specially xylene twice, 100% ethanol: xylene is 1: 2 solution, 100% ethanol: xylene is 1: 1 solution, 100% ethanol: xylene is 2: 1 solution, 100% ethanol twice, afterwards with concentration ethanolic solution rehydration from high to low.
6) dyeing time confirm as sarranine dyeing 2～4h, fast green dyeing 35～45s because blade heavy leather matter, dyeing time is got good result than normal dyeing time lengthening.
7) replace neutral gum with transparent nail polish and carry out mounting, soaked the tissue of nail polish, tissue staining is especially clearly demarcated, and is clear.
Description of drawings
Fig. 1 is Cuba's cow's milk tree (Manilkara roxburghiana (Wight) Dubard) blade construction.In Fig. 1, scale is 200 μ m.
Fig. 2 is Cuba's cow's milk tree (Manilkara roxburghiana (Wight) Dubard) vascular bundle structure.
Fig. 3 is Cuba's cow's milk tree (Manilkara roxburghiana (Wight) Dubard) blade construction.
Fig. 4 is Cuba's cow's milk tree (Manilkara roxburghiana (Wight) Dubard) blade construction.
Fig. 5 is miracle fruit (Synsepalum dulcifcum Denill) blade construction.In Fig. 5, scale is 100 μ m.
Fig. 6 is chrysophyllum cainito (Chrysophyllum cainito L.) blade construction.In Fig. 6, scale is 100 μ m.
In Fig. 1～6, respectively be labeled as: 1. palisade tissue; 2. vascular bundle; 3. spongy tissue; 4. conduit; 5. screen casing; 6. conduit; 7. palisade tissue; 8. mesophyll tissue; 9. conduit; 10. palisade tissue; 11. spongy tissue.
Embodiment
The paraffin section instance:
Embodiment 1
Concrete steps are following:
1) gets fresh disease-free Cuba cow's milk tree (Manilkara roxburghiana (Wight) Dubard) blade (thickness 450～500 μ m; Keratin); With single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2) fixing: fixing agent adopts omnipotent immobile liquid (also claim the FAA immobile liquid, prescription is 70% ethanol 90ml, glacial acetic acid 5ml, formalin 5ml), set time 24h, but and long preservation.
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 4h is specially 4h in 4h in 4h in 4h in 4h in 4h in 4h in 60% ethanol 4h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 4h (for the first time) → 100% ethanol 4h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 3h, detailed process is an xylene: 100% ethanol is in solution to soak 3h → xylene at 1: 2: 100% ethanol is in solution to soak 3h → xylene at 1: 1: 100% ethanol is in solution to soak in 3h → pure xylene soak at 2: 1 to soak 3h (for the second time) in 3h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 48h in 37 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 60 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6) section: adopt common biological microtome section, slice thickness 10～12 μ m.
7) exhibition sheet: earlier in 30% ethanol transition, the back is to open up sheet in the warm water in 50 ℃ in water temperature.
8) paster: do not use the bonding die agent, directly slightly dry by the fire sheet with ethanol lamp flame envelope.
9) roasting sheet: roasting sheet 30h in 37 ℃ incubator.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 1.5min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 1.5min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 1.5min → 100% ethanol 1.5min (for the first time) → 100% ethanol 1.5min (for the second time) → 95% ethanol 1.5min → 85% ethanol, 1～2min → 70% ethanol 1.5min → 50% ethanol 1.5min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 3h → 95% ethanol 1.5min → 100% ethanol 1.5min (for the first time) → 100% ethanol 1.5min (for the second time) 1.5min → 100% ethanol: xylene is 1.5min → 100% ethanol in 2: 1 solution: xylene is 1.5min → 100% ethanol in 1: 1 solution: xylene is 1.5min → xylene 12min (for the first time) → fast green 40s that redyes of xylene 12min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting (the section effect is seen accompanying drawing 1) with transparent nail polish.
1)～4) as describing among the embodiment 1.
5) passing through wax, to adopt fusing point be 52～54 ℃ soft wax, and adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 72h in 37 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 63 ℃ of constant temperature ovens afterwards into, during to change one to twice paraffin refined wax, embedding afterwards.
6) as describing among the embodiment 1.
7) exhibition sheet: earlier in 25% ethanol transition, the back is to open up sheet in the warm water in 55 ℃ in water temperature.
8) as describing among the embodiment 1.
9) roasting sheet: roasting sheet 36h in 35 ℃ incubator.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 1min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 1min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 1min → 100% ethanol 1min (for the first time) → 100% ethanol 1min (for the second time) → 95% ethanol 1min → 85% ethanol 1min → 70% ethanol 1min → 50% ethanol 1min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 3h → 95% ethanol 1min → 100% ethanol 1min (for the first time) → 100% ethanol 1min (for the second time) → 100% ethanol: xylene is 1min → 100% ethanol in 2: 1 solution: xylene is 1min → 100% ethanol in 1: 1 solution: xylene is 1min → xylene 10min (for the first time) → fast green 35s that redyes of xylene 10min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting (the section effect is seen accompanying drawing 2) with Canadian neutral gum.
1)～6) as describing among the embodiment 1.
7) exhibition sheet: earlier in 35% ethanol transition, the back is to open up sheet in the warm water in 48 ℃ in water temperature.
8) as describing among the embodiment 1.
9) roasting sheet: roasting sheet 36h in 35 ℃ incubator.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 2min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 2min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) → 95% ethanol 2min → 85% ethanol 2min → 70% ethanol 2min → 50% ethanol 2min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 3h → 95% ethanol 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) → 100% ethanol: xylene is 2min → 100% ethanol in 2: 1 solution: xylene is 2min → 100% ethanol in 1: 1 solution: xylene is 2min → xylene 15min (for the first time) → fast green 35s that redyes of xylene 15min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting (the section effect is seen accompanying drawing 3) with transparent nail polish.
Concrete steps are following:
1)～2) as describing among the embodiment 1.
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 4h is specially 4h in 4h in 4h in 4h in 4h in 4h in 4h in 60% ethanol 4h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 4h (for the first time) → 100% ethanol 4h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 2h, detailed process is an xylene: 100% ethanol is in solution to soak 2h → xylene at 1: 2: 100% ethanol is in solution to soak 2h → xylene at 1: 1: 100% ethanol is in solution to soak in 2h → pure xylene soak at 2: 1 to soak 2h (for the second time) in 2h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 72h in 37 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 63 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6)～12) as describing (the section effect is seen accompanying drawing 4) among the embodiment 1.
Concrete steps are following:
1)～2) as describing among the embodiment 1.
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 5h is specially 5h in 5h in 5h in 5h in 5h in 5h in 5h in 60% ethanol 5h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 5h (for the first time) → 100% ethanol 5h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 4h, detailed process is an xylene: 100% ethanol is in solution to soak 4h → xylene at 1: 2: 100% ethanol is in solution to soak 4h → xylene at 1: 1: 100% ethanol is in solution to soak in 4h → pure xylene soak at 2: 1 to soak 4h (for the second time) in 4h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 60h in 40 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 60 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6)～12) as describing among the embodiment 1.
Concrete steps are following:
1)～2) as describing among the embodiment 1.
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 5h is specially 5h in 5h in 5h in 5h in 5h in 5h in 5h in 60% ethanol 5h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 5h (for the first time) → 100% ethanol 5h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 4h, detailed process is an xylene: 100% ethanol is in solution to soak 4h → xylene at 1: 2: 100% ethanol is in solution to soak 4h → xylene at 1: 1: 100% ethanol is in solution to soak in 4h → pure xylene soak at 2: 1 to soak 4h (for the second time) in 4h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 72h in 40 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 63 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6)～9) as describing among the embodiment 1.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 2min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 2min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) → 95% ethanol 2min → 85% ethanol 2min → 70% ethanol 2min → 50% ethanol 2min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 3h → 95% ethanol 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) → 100% ethanol: xylene is 2min in 21 solution → 100% ethanol: xylene is 2min → 100% ethanol in 1: 1 solution: xylene is 2min → xylene 12min (for the first time) → fast green 40s that redyes of xylene 12min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting with Canadian neutral gum.
Concrete steps are following:
1)～2) as describing among the embodiment 1.
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 5h is specially 5h in 5h in 5h in 5h in 5h in 5h in 5h in 60% ethanol 5h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 5h (for the first time) → 100% ethanol 5h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 4h, detailed process is an xylene: 100% ethanol is in solution to soak 4h → xylene at 1: 2: 100% ethanol is in solution to soak 4h → xylene at 1: 1: 100% ethanol is in solution to soak in 4h → pure xylene soak at 2: 1 to soak 4h (for the second time) in 4h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 72h in 40 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 63 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6)～9) as describing among the embodiment 1.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 2min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 2min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) → 95% ethanol 2min → 85% ethanol 2min → 70% ethanol 2min → 50% ethanol 2min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 3h → 95% ethanol 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) → 100% ethanol: xylene is 2min → 100% ethanol in 2: 1 solution: xylene is 2min → 100% ethanol in 1: 1 solution: xylene is 2min → xylene 10min (for the first time) → fast green 45s that redyes of xylene 10min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting with Canadian neutral gum.
Concrete steps are following:
1)～2) as describing among the embodiment 1.
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 3h is specially 3h in 3h in 3h in 3h in 3h in 3h in 3h in 60% ethanol 3h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 3h (for the first time) → 100% ethanol 3h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 2h, detailed process is an xylene: 100% ethanol is in solution to soak 2h → xylene at 1: 2: 100% ethanol is in solution to soak 2h → xylene at 1: 1: 100% ethanol is in solution to soak in 2h → pure xylene soak at 2: 1 to soak 2h (for the second time) in 2h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 60h in 37 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 60 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6)～9) as describing among the embodiment 1.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 1min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 1min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 1min → 100% ethanol 1min (for the first time) → 100% ethanol 1min (for the second time) → 95% ethanol 1min → 85% ethanol 1min → 70% ethanol 1min → 50% ethanol 1min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 3h → 95% ethanol 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) → 100% ethanol: xylene is 2min → 100% ethanol in 2: 1 solution: xylene is 2min → 100% ethanol in 1: 1 solution: xylene is 2min → xylene 12min (for the first time) → fast green 38s that redyes of xylene 12min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting with transparent nail polish.
Embodiment 9
Concrete steps are following:
1)～2) of embodiment 1
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 5h is specially 5h in 5h in 5h in 5h in 5h in 5h in 5h in 60% ethanol 5h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 5h (for the first time) → 100% ethanol 5h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 3h, detailed process is an xylene: 100% ethanol is in solution to soak 3h → xylene at 1: 2: 100% ethanol is in solution to soak 3h → xylene at 1: 1: 100% ethanol is in solution to soak in 3h → pure xylene soak at 2: 1 to soak 3h (for the second time) in 3h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 48h in 37 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 60 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6) section: adopt common biological microtome section, slice thickness 10～12 μ m.
7) exhibition sheet: earlier in 25% ethanol transition, the back is to open up sheet in the warm water in 50 ℃ in water temperature.
8) paster: do not use the bonding die agent, directly slightly dry by the fire sheet with ethanol lamp flame envelope.
9) roasting sheet: roasting sheet 36h in 35 ℃ incubator.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 1.5min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 1.5min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 1.5min → 100% ethanol 1.5min (for the first time) → 100% ethanol 1.5min (for the second time) → 95% ethanol 1.5min → 85% ethanol, 1～2min → 70% ethanol 1.5min → 50% ethanol 1.5min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 4h → 95% ethanol 1min → 100% ethanol 1min (for the first time) → 100% ethanol 1min (for the second time) 1min → 100% ethanol: xylene is 1min → 100% ethanol in 2: 1 solution: xylene is 1min → 100% ethanol in 1: 1 solution: xylene is 1min → xylene 12min (for the first time) → fast green 40s that redyes of xylene 12min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting with transparent nail polish.
Embodiment 10
Concrete steps are following:
1)～2) of embodiment 1
3) dehydration: adopt ethanol dehydration; Begin with 60% ethanol; 5% increases progressively; Every grade of 3h is specially 3h in 3h in 3h in 3h in 3h in 3h in 3h in 60% ethanol 3h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol 3h (for the first time) → 100% ethanol 3h (for the second time).
4) transparent: as to take different proportion (volume ratio) solution of xylene and ethanol to do transition; Every grade of 3h, detailed process is an xylene: 100% ethanol is in solution to soak 3h → xylene at 1: 2: 100% ethanol is in solution to soak 3h → xylene at 1: 1: 100% ethanol is in solution to soak in 3h → pure xylene soak at 2: 1 to soak 3h (for the second time) in 3h (for the first time) → pure xylene.
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly; Adopt xylene: paraffin is 1: 1 (volume ratio) solution transition waxdip 72h in 40 ℃ of constant temperature ovens, changes paraffin refined wax waxdip in 63 ℃ of constant temperature ovens afterwards into, during to change paraffin refined wax one to twice; Use template after passing through wax; Three is a thickness, and the big carton of folding 2cm * 2cm * 3cm is used for embedding.
6) section: adopt common biological microtome section, slice thickness 10～12 μ m.
7) exhibition sheet: earlier in 35% ethanol transition, the back is to open up sheet in the warm water in 48 ℃ in water temperature.
8) paster: do not use the bonding die agent, directly slightly dry by the fire sheet with ethanol lamp flame envelope.
9) roasting sheet: roasting sheet 30h in 37 ℃ incubator.
10) dewaxing rehydration: program is specially 100% ethanol: xylene is 1.5min → 100% ethanol in 1: 2 (volume ratio) solution: xylene is 1.5min → 100% ethanol in 1: 1 (volume ratio) solution: xylene is 1.5min → 100% ethanol 1.5min (for the first time) → 100% ethanol 1.5min (for the second time) → 95% ethanol 1.5min → 85% ethanol, 1～2min → 70% ethanol 1.5min → 50% ethanol 1.5min in 2: 1 (volume ratio) solution.
11) dyeing: specific procedure is 1% sarranine solution-dyed 2h → 95% ethanol 2min → 100% ethanol 2min (for the first time) → 100% ethanol 2min (for the second time) 2min → 100% ethanol: xylene is 2min → 100% ethanol in 2: 1 solution: xylene is 2min → 100% ethanol in 1: 1 solution: xylene is 2min → xylene 10min (for the first time) → fast green 40s that redyes of xylene 10min (for the second time) → 1% in 1: 2 solution.
12) mounting: carry out mounting with Canadian neutral gum.
Embodiment 11
Concrete steps are following:
1) get fresh disease-free miracle fruit (Synsepalum dulcifcum Denill) blade (thickness 100～130 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) like embodiment 1 said (the section effect is seen accompanying drawing 5).
Embodiment 12
Concrete steps are following:
1) get fresh disease-free miracle fruit (Synsepalum dulcifcum Denill) blade (thickness 100～130 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 3.
Embodiment 13
Concrete steps are following:
1) get fresh disease-free miracle fruit (Synsepalum dulcifcum Denill) blade (thickness 100～130 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 5.
Embodiment 14
Concrete steps are following:
1) get fresh disease-free miracle fruit (Synsepalum dulcifcum Denill) blade (thickness 100～130 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 7.
Embodiment 15
Concrete steps are following:
1) get fresh disease-free miracle fruit (Synsepalum dulcifcum Denill) blade (thickness 100～130 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 9.
Embodiment 16
Concrete steps are following:
1) get fresh disease-free chrysophyllum cainito (Chrysophyllum cainito L.) blade (thickness 200～250 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) like embodiment 2 said (the section effect is seen accompanying drawing 6).
Embodiment 17
Concrete steps are following:
1) get fresh disease-free chrysophyllum cainito (Chrysophyllum cainito L.) blade (thickness 200～250 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 4.
Embodiment 18
Concrete steps are following:
1) get fresh disease-free chrysophyllum cainito (Chrysophyllum cainito L.) blade (thickness 200～250 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 6.
Embodiment 19
Concrete steps are following:
1) get fresh disease-free chrysophyllum cainito (Chrysophyllum cainito L.) blade (thickness 200～250 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 8.
Embodiment 20
Concrete steps are following:
1) get fresh disease-free chrysophyllum cainito (Chrysophyllum cainito L.) blade (thickness 200～250 μ m, papery), with single-edge blade each 5～6mm place incision along the master pulse both sides, with the nervate part of central authorities again crosscut become the segment of 5～6mm.
2)～12) of embodiment 10.
Claims (8)
1. heavy leather matter blade paraffin section method is characterized in that may further comprise the steps:
1) with blade along master pulse two lateral incisions, with the nervate part of central authorities again crosscut become segment;
2) the resulting blade of step 1) is fixed with fixing agent;
3) with step 2) with the fixing blade dehydration of fixing agent; The concrete grammar of said dehydration is: begin with 60% ethanol; 5% increases progressively; Every grade of 3～5h is specially 3～5h in 3～5h in 3～5h in 3～5h in 3～5h in 3～5h in 3～5h in 60% ethanol, 3～5h → 65% ethanol → 70% ethanol → 75% ethanol → 80% ethanol → 85% ethanol → 90% ethanol → 95% ethanol → 100% ethanol, 3～5h → 100% ethanol, 3～5h;
4) do transition with the solution of the different volumes ratio of xylene and ethanol; Every grade of 2～4h, detailed process is an xylene: 100% ethanol is in solution to soak 2～4h → xylene at 1: 2: 100% ethanol is in solution to soak 2～4h → xylene at 1: 1: 100% ethanol is in solution to soak in 2～4h → pure xylene soak in 2～4h → pure xylene at 2: 1 to soak 2～4h;
5) pass through wax and embedding: wax employing fusing point is 52～54 ℃ a soft wax thoroughly, and by volume, xylene: paraffin is 1: 1 solution transition waxdip 48～72h in 37～40 ℃ of constant temperature ovens; Change paraffin refined wax waxdip in 60～63 ℃ of constant temperature ovens afterwards into; Will change paraffin refined wax during this time 1 or 2 time, use template behind the wax thoroughly, 3 is a thickness; The big carton of folding 2cm * 2cm * 3cm is used for embedding;
6) section back exhibition sheet, paster is baked sheet then again;
7) dewaxing rehydration; The concrete grammar of said dewaxing rehydration is: by volume, 100% ethanol: xylene is 1～2min → 100% ethanol in 1: 2 solution: xylene is 1～2min → 100% ethanol in 1: 1 solution: xylene is 1～2min → 100% ethanol, 1～2min → 100% ethanol, 1～2min → 95% ethanol, 1～2min → 85% ethanol, 1～2min → 70% ethanol, 1～2min → 50% ethanol, 1～2min in 2: 1 solution;
8) dyeing back mounting.
2. heavy leather matter blade paraffin section method as claimed in claim 1 is characterized in that in step 1), said with blade along master pulse two lateral incisions, be with single-edge blade each 5～6mm place incision along the master pulse both sides; The length of said segment is 5～6mm.
3. heavy leather matter blade paraffin section method as claimed in claim 1 is characterized in that in step 2) in, said fixing agent is omnipotent immobile liquid, prescription is 70% ethanol 90ml, glacial acetic acid 5ml, formalin 5ml; Said regular time is 24h at least.
4. heavy leather matter blade paraffin section method as claimed in claim 1 is characterized in that in step 6), and the section of biologic slice machine is adopted in said section, and the thickness of section is 10～12 μ m.
5. heavy leather matter blade paraffin section method as claimed in claim 1 is characterized in that in step 6), and said exhibition sheet is first in 25%～35% ethanol transition, is to open up sheet in the warm water in 48～55 ℃ in water temperature then.
6. heavy leather matter blade paraffin section method as claimed in claim 1 is characterized in that in step 6) said paster is directly slightly to dry by the fire sheet with ethanol lamp flame envelope.
7. heavy leather matter blade paraffin section method as claimed in claim 1 is characterized in that in step 6), and said roasting sheet is roasting sheet 24～36h in 35～39 ℃ incubator.
8. heavy leather matter blade paraffin section method as claimed in claim 1; It is characterized in that in step 8) the concrete grammar of said dyeing is: 1% sarranine solution-dyed, 2～4h → 95% ethanol, 1～2min → 100% ethanol, 1～2min → 100% ethanol, 1～2min → 100% ethanol: xylene is 1～2min → 100% ethanol in 2: 1 solution: xylene is 1～2min → 100% ethanol in 1: 1 solution: xylene is the fast green 35～45s that redyes of 1～2min → xylene 10～15min → xylene 10～15min → 1% in 1: 2 solution; Said mounting adopts canada balsam or transparent nail polish to carry out mounting.
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| CN102607907B (en) * | 2012-02-24 | 2014-08-27 | 东北农业大学 | Paraffin section method for fern gametophytes |
| CN102967493B (en) * | 2012-10-27 | 2015-04-08 | 山西农业大学 | Rapid paraffin sectioning method for plant tissue |
| CN102967497B (en) * | 2012-12-05 | 2015-09-30 | 沈阳农业大学 | Position observation in plant leaf blade micromechanism and the method that blade is processed |
| CN104614211B (en) * | 2015-01-19 | 2017-11-03 | 沈阳农业大学 | A kind of Tiannu Magnolia seed paraffin section method |
| CN104849110B (en) * | 2015-04-03 | 2018-03-16 | 成都大学 | A kind of paraffin section method of plant tissue |
| CN105651573B (en) * | 2016-01-05 | 2018-05-01 | 中国科学院华南植物园 | One Leaf Epidermis hair removal methods |
| CN105973673B (en) * | 2016-06-30 | 2018-01-02 | 中国林业科学研究院热带林业研究所 | A kind of paraffin section method of eucalyptus tissue |
| CN106501053B (en) * | 2016-12-30 | 2019-02-26 | 广西壮族自治区药用植物园 | The paraffin section method of meadowrueleaf corydalis root blade |
| CN106706389B (en) * | 2016-12-30 | 2019-07-05 | 广西壮族自治区药用植物园 | The paraffin section method of meadowrueleaf corydalis root root |
| CN108844796A (en) * | 2018-06-29 | 2018-11-20 | 四川农业大学 | A kind of blade paraffin section method |
| CN111948008A (en) * | 2020-07-14 | 2020-11-17 | 中国科学院华南植物园 | Paraffin penetrating method for paraffin section |
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| CN101324493A (en) * | 2008-07-29 | 2008-12-17 | 孙翔宇 | Method for rapidly manufacturing large tissue specimen slice |
| CN101762412B (en) * | 2009-12-30 | 2011-07-20 | 西南大学 | Soap embedded plant section method |
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