CN106546473A - It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud - Google Patents
It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud Download PDFInfo
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- CN106546473A CN106546473A CN201611056532.2A CN201611056532A CN106546473A CN 106546473 A CN106546473 A CN 106546473A CN 201611056532 A CN201611056532 A CN 201611056532A CN 106546473 A CN106546473 A CN 106546473A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
- G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy
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Abstract
The present invention relates to a kind of embed flaking method for Caulis Sacchari sinensis Different node lateral bud, belong to plant tissue embedded section technical field, including（1）Sample draw materials it is fixed,（2）Soften,（3）Dehydration,（4）Displacement,（5）Infiltration,（6）Embedding,（7）Repair block,（8）Section,（9）Exhibition piece, bonding die,（10）The steps such as dyeing；The characteristics of the method synthesis paraffin section embedding and resin penetration embed two kinds of embedded section methods, integrate application, learn from other's strong points to offset one's weaknesses, research and develop the section that novel system ground solves the problems, such as Caulis Sacchari sinensis lateral bud cytology research at present, and the section made is complete, almost do not crush, can reach the section embedding effect for taking into account entirety and details.
Description
Technical field
The present invention relates to a kind of embed flaking method for Caulis Sacchari sinensis Different node lateral bud, belong to plant tissue embedded section skill
Art field.
Background technology
Plant tissue embedded section technology is the most frequently used a kind of technology in organic section making technology.Due to its can be cut into compared with
It is thin and continuously cut into slices, plant anatomy scholar mainly organized with its observation of cell and organ form, make Permanent slide preservation.
What embedded section technology was the most commonly used is that paraffin section embedding and resin penetration are embedded.Wherein paraffin section embedding step includes taking
Material, fixation, dehydration, transparent, waxdip, embedding, section, dewaxing, transparent, dyeing etc., resin slicer embedding step include drawing materials, consolidate
Fixed, rear fixation, dehydration, displacement, resin penetration, embedding, section and dyeing etc..Using both the above method, have some and be directed to
The embedded section technology of different plant tissue exploitations.Such as a kind of Garnetophytes development paraffin of CN201210043067.4 exploitations
The method of section, CN201210408507.1 provide a kind of paraffin section method and its paper of peanut kernel《Resin embedding skill
Application of the art in the section of fibre section》On the basis of the characteristic of acrylic resin embedding medium, have studied a kind of based on pre-polymerization
Resin Rapid embedding technology of technology, etc..
For Caulis Sacchari sinensis research of agricultural science person, the development of Caulis Sacchari sinensis sections lateral bud be need research important object it
One, tradition is used mostly paraffin section and is studied, and also has foreign literature to show a small amount of thin using cutting half after epoxy resin embedding
Section is studied.For the former, although technology maturation, can cut compared with large sample, but there is fixative to sample damage
It is excessive, cut into slices thicker（8μm）, the shortcoming that definition in blocks is not high and the time is tediously long.Latter resins' section is belonged to originally in transmission electron microscope
The category of ultrathin section, its light microscopic semithin section are only used for the positioning means of its electron microscopyc sample preparation, fix liquid temperature yet with sample
With, more slight is affected on histiocyte deformation etc., section can be very thin（0.5~1μm）, coordinate appropriate dyeing liquor, clearly
Degree can exceed paraffin section a lot, but have the shortcomings that cut sample can not be excessive.
Further, since growth of the Caulis Sacchari sinensis lateral bud with joint number, the wooden bolt matterization of lateral bud cells of superficial layer near base portion is serious,
Often because section is broken when being cut into slices after prior paraffin embedding, Cavitated and satisfied result cannot be obtained.In summary,
Caulis Sacchari sinensis lateral bud cytology research has Sample location, repaiies block trouble, fixed incomplete, frangible, lack of defination in blocks of cutting into slices,
Embedding medium is wasted, it is impossible to which high-volume sample preparation is for the problem of follow-up measurement analysis.
The content of the invention
The problem that the present invention is present for prior art, there is provided a kind of to embed film-maker for Caulis Sacchari sinensis Different node lateral bud
The characteristics of method, comprehensive paraffin section embedding and resin penetration embed two kinds of embedded section methods, integrates application, learns from other's strong points to offset one's weaknesses, with
The section of Caulis Sacchari sinensis lateral bud cytology research is solved the problems, such as at present systematically.
In order to solve the above problems, the technical solution adopted in the present invention is：
It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud, comprise the following steps：
（1）Sample is drawn materials fixation：Double-edged razor blade extracts the fresh lateral bud rip cutting plucked in each stem section of Caulis Sacchari sinensis, for ending starts the
The bud of a piece of full lamina piece correspondence sections calculates No. 1, is cut downwards according to Research Requirements successively, and sub-bottle is fixed, and per bottle is put into about 2ml
Previous ready 2.5% glutaraldehyde fixative；
（2）Soften：After previous step about 4h, for the sample near top（About 1 ~ No. 4 lateral bud）Select to continue to fix, remaining side
Fixative is extracted out by bud, buffer solution for cleaning three times, per minor tick 10min, is sucked buffer afterwards, is put into 4% ethylenediamine water-soluble
Liquid, stands 8 ~ 10h and softens at 4 DEG C, can 30 ~ 60 min of proper extension set time if close base portion lateral bud is really up to the mark；
（3）Dehydration：By the lateral bud after softening/not through bating step tender shoots in the lump with buffer solution for cleaning three times, every time between
Every 10min, buffer is sucked afterwards, improve 20%, 40%, 60%, 80%, 100% step by step according to alcohol concentration and be dehydrated, often walk
20 ~ 25min, if time of day is too late, can directly switch to 70% ethanol overnight after 60% ethanol, the next day continue.To ensure dehydration
Thoroughly, 100% ethanol needs three times, each 20min；
（4）Displacement：
A. for the lateral bud for numbering larger close base portion（Harder, volume is larger）, take paraffin section embedding flow process, displacement
Step adopts dimethylbenzene, first removes absolute alcohol, adds dimethylbenzene/ethanol 1:1 mixed liquor, stands 30min, pours out afterwards and use 2 instead:
1 mixed liquor, uses pure dimethylbenzene instead after 30min；
B. for the lateral bud of tenderer close ending（It is softer, small volume）, take resin slicer embedding flow process, displacement step
Using expoxy propane, first go in absolute alcohol, sample bottle, to add expoxy propane/ethanol 1:1 mixed liquor, stands 10min, falls afterwards
Go out to use 2 instead:1 mixed liquor, uses pure propylene oxide instead after 10min, a pure propylene oxide is changed per 10min, in triplicate；
（5）Infiltration：
A. for paraffin step,, to ooze wax process, the pure dimethylbenzene of previous step adds the paraffin of fusing for this, and temperature is 65 ~
75 DEG C, 20 ~ 30min of process time changes the paraffin of fusing, reprocesses 20 ~ 30min, obtains the lateral bud tissue of waxdip
B., for resin embedding step, it is 1ml by expoxy propane restricted volume in 5.2 steps each sample bottle, then
Deca 0.2ml epoxy resin, mixes, and stands 2h, afterwards Deca 0.2ml resin per hour, improves three mistakes of resin concentration step by step
Afterwards, half mixed liquor is sucked, equal proportion adds second half virgin resin, mixes and stands 3h, changes virgin resin 12h infiltrations, overall process is extremely
Gently shake to promote embedding medium to permeate in low speed shaking table
（6）Embedding：Using the detachable embedding plate of special design；
A. for paraffin section, using increasing plate；
B. for resin slicer, using basic plate；
（7）Repair block：Save, or only need slightly to grind off a little unnecessary paraffin/resin of lateral bud sample periphery；
（8）Section：
For wax embedding block, base just directly can be cut into slices with the bonding of amonang block, 8 μm of slice thickness；Resin embedding flow process
Sample blocks, its base just can be cut into slices using ultramicrotome on diamant with pedestal resin-bonding, 1 μm of slice thickness
（9）Exhibition piece, bonding die：
A. the wax band comprising sample is gently provoked after slice, thin piece is cut out by paraffin-embedded sample using brush pen, is placed in advance
On the microscope slide of washed and Deca few drops distilled water, 50 DEG C of exhibition pieces, roasting piece 30min are brought into close contact microscope slide up to section, subsequently
Insert to stand in pure dimethylbenzene dewaxing cylinder and slough within ten minutes paraffin, tweezers gently take out the microscope slide after dewaxing, are sequentially placed into
100%th, 90%, 70%, 50%, 30%, 10% ethanol enters line replacement, and last microscope slide Deca distilled water carries out rinse, bakes piece to be dyed；
B., for resin slicer, the slice, thin piece of 1 μ m-thick for directly cutting ultramicrotome is using specific purpose tool from tank
Choose and be placed on the microscope slide of few drops distilled water, 70 DEG C are dried；
（10）Dyeing
Paraffin section conventionally sarranine Fast Green counterstain, the TBO stains that can also use resin slicer to commonly use；Resin slicer
Using staining counter, the slice, thin piece that previous step is glued（Microscope slide）It is placed on roasting piece platform, 40 DEG C, respectively using the process of 1% sarranine
1min, after cleaning, 0.5%TBO processes 1min, and last 0.5% crystal violet is processed, and distilled water bakes piece after cleaning.
Step（1）2.5% described glutaraldehyde fixative, with 0.05mol/L pH7.2 phosphate buffers or 0.05mol/L
Cacodylic acid buffer, latter of which are better, and add 1% sucrose, 4 DEG C of preservations after preparation；
Step（5）Described epoxy resin, its formula are resin embedding host, plasticiser, sclerosing agent, catalyst amount（Quality）
Than for 10:5.5:26:0.4.
The invention has the beneficial effects as follows：
（1）The characteristics of synthesis paraffin section of the invention embedding and resin penetration embed two kinds of embedded section methods, integrates application, takes
Long benefit is short, for the Caulis Sacchari sinensis lateral bud rip cutting sample near morphology upper end, takes resin embedding flow process, for middle and lower part sections
Sample takes the fixation of resin slicer flow process then to switch to paraffin embedding and flow process of cutting into slices, while adding to the two pretreatment process soft
Change step, lower the broken probability of later stage section, and using special design mould, systematically solve current Caulis Sacchari sinensis lateral bud cell
Learn the section problem of research, from tender to old, a collection of solution from top to bottom, with good scientific research guidance and Practical significance.
（2）By the abundant research organized to Caulis Sacchari sinensis lateral bud and lot of experimental data, during the present invention is to embedded section
Targeted design is carried out with using material：A. 1% sucrose is added to increase the osmosiss of fixative in fixative so that Gu
Constant speed degree faster, and is not adopted in conventional sample preparation；B. the fixative of routine paraffin wax embedding is fixed for FAA or Carnoy ' s
Liquid, the present invention innovatively use the improved glutaraldehyde fixation that flow process is embedded based on resin slicer, before can so reducing
The trouble that phase is processed, it is often more important that glutaraldehyde has more preferable effect for fixed cell protein, and to being subsequently used for paraffin
Section is also significantly improved；C. the Formulaion of epoxy resin for adopting is otherwise varied compared with tradition, conventional Spurr resin embeddings host,
Plasticiser, sclerosing agent, catalyst amount（Quality）Than for 10:6:26:0.4, and the DER736 plasticisers ratio of the present invention is reduced
To 5.5, suitably to lower the hardness after embedded block polymerization, to be conducive to diamant semithin section, sample not to allow rapid wear knife, produce
Tool marks.
（3）The section made using the present invention is complete, does not almost crush, and most hard perula also intactly can protect very much
Deposit；The structures such as vascular bundle preserve very intact, meanwhile, individual cells content is well secured display, nucleus, albumen
Body, vacuole are high-visible, provide guarantee for follow-up works such as the cell counting under subsequent image acquisition, light microscopic, area measurements.
The present invention can reach the section embedding effect for taking into account entirety and details, compensate for embedding using prior paraffin and resin
The brought shortcoming of embedding.
Description of the drawings
Resin slicer rip cutting figures of the Fig. 1 for Section 5 section lateral bud, shoots under 10 × object lens；
Fig. 2 is ending（Section 1 section）The resin slicer rip cutting figure of lateral bud, 10 × object lens shoot；
Resin slicer rip cutting figures of the Fig. 3 for Section 10 section lateral bud, shoots under 10 × object lens；
Fig. 4 be Section 10 section lateral bud resin slicer rip cutting perula cells of superficial layer local detail, the visible cell Jing after the flow processing
Interior details is clear, does not have breakage, 100 × object lens（Under oil mirror）；
Fig. 5 is the local detail of cell above Section 10 section lateral bud resin slicer rip cutting growth cone, it is seen that cellular content（Liquid
Bubble, nucleus and kernel etc.）It is very clear, 100 × object lens（Under oil mirror）；
The local detail of cell below Fig. 6 Section 10 section lateral bud resin slicer rip cutting growth cones, it is seen that various types of cells and intracellular
It is tolerant very clear, 100 × object lens（Under oil mirror）；
Fig. 7 is the paraffin section rip cutting figure of middle part lateral bud（Perula local）, 5 × object lens；
Fig. 8 is middle and lower part lateral bud（Section 15 section）Paraffin section rip cutting figure（Perula local）, 5 × object lens；
Fig. 9 is prior paraffin Sectioning（Without bating step）The stage casing lateral bud longitudinal section made, it is seen that section is broken；
Figure 10 be paraffin section figure, the most hard lateral bud of base portion（20+ sections）, 5 × object lens magnification.
Specific embodiment
The present invention is described in further details below by embodiment, these embodiments are only used for illustrating the present invention, and
Do not limit the scope of the invention.
Embodiment 1
It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud, comprise the following steps：
（1）Sample is drawn materials fixation：Double-edged razor blade extracts the fresh lateral bud rip cutting plucked in each stem section of Caulis Sacchari sinensis, for ending starts the
The bud of a piece of full lamina piece correspondence sections calculates No. 1, is cut downwards according to Research Requirements successively, and sub-bottle is fixed, and per bottle is put into about 2ml
Previous ready 2.5% glutaraldehyde fixative；
（2）Soften：After previous step about 4h, for the sample near top（About 1 ~ No. 4 lateral bud）Select to continue to fix, remaining side
Fixative is extracted out by bud, buffer solution for cleaning three times, per minor tick 10min, is sucked buffer afterwards, is put into 4% ethylenediamine water-soluble
Liquid, stands 8 h softenings at 4 DEG C, can 30 min of proper extension set time if close base portion lateral bud is really up to the mark；
（3）Dehydration：By the lateral bud after softening/not through bating step tender shoots in the lump with buffer solution for cleaning three times, every time between
Every 10min, buffer is sucked afterwards, improve 20%, 40%, 60%, 80%, 100% step by step according to alcohol concentration and be dehydrated, often walk
20min, if time of day is too late, can directly switch to 70% ethanol overnight after 60% ethanol, the next day continue.It is thorough to ensure dehydration
Bottom, 100% ethanol need three times, each 20min；
（4）Displacement：
4.1 for the lateral bud for numbering larger close base portion（Harder, volume is larger）, take paraffin section embedding flow process, displacement
Step adopts dimethylbenzene, first removes absolute alcohol, adds dimethylbenzene/ethanol 1:1 mixed liquor, stands 30min, pours out afterwards and use 2 instead:
1 mixed liquor, uses pure dimethylbenzene instead after 30min；
4.2 for the lateral bud of tenderer close ending（It is softer, small volume）, take resin slicer embedding flow process, displacement step
Using expoxy propane, first go in absolute alcohol, sample bottle, to add expoxy propane/ethanol 1:1 mixed liquor, stands 10min, falls afterwards
Go out to use 2 instead:1 mixed liquor, uses pure propylene oxide instead after 10min, a pure propylene oxide is changed per 10min, in triplicate；
（5）Infiltration：
5.1. for paraffin step,, to ooze wax process, the pure dimethylbenzene of previous step adds the paraffin of fusing for this, and temperature is 65 ~
75 DEG C, process time 20min changes the paraffin of fusing, reprocesses 20min, obtains the lateral bud tissue of waxdip
5.2 for resin embedding step, is 1ml by expoxy propane restricted volume in 5.2 steps each sample bottle, then
Deca 0.2ml epoxy resin, mixes, and stands 2h, afterwards Deca 0.2ml resin per hour, improves three mistakes of resin concentration step by step
Afterwards, half mixed liquor is sucked, equal proportion adds second half virgin resin, mixes and stands 3h, changes virgin resin 12h infiltrations, overall process is extremely
Gently shake to promote embedding medium to permeate in low speed shaking table
（6）Embedding：Using the detachable embedding plate of special design；
For 6.1 paraffin sections, using increasing plate；
For 6.2 resin slicers, using basic plate；
（7）Repair block：Save, or only need slightly to grind off a little unnecessary paraffin/resin of lateral bud sample periphery；
（8）Section：
For wax embedding block, base just directly can be cut into slices with the bonding of amonang block, 8 μm of slice thickness；Resin embedding flow process
Sample blocks, its base just can be cut into slices using ultramicrotome on diamant with pedestal resin-bonding, 1 μm of slice thickness
（9）Exhibition piece, bonding die：
Wax band comprising sample after slice, thin piece is cut out gently is provoked using brush pen, is placed on pre- by 9.1 paraffin-embedded samples
First wash and on the microscope slide of Deca few drops distilled water, 50 DEG C of exhibition pieces, roasting piece 30min are brought into close contact microscope slide up to section, with
After insert to stand in pure dimethylbenzene dewaxing cylinder and slough within ten minutes paraffin, tweezers gently take out the microscope slide after dewaxing, are sequentially placed into
100%th, 90%, 70%, 50%, 30%, 10% ethanol enters line replacement, and last microscope slide Deca distilled water carries out rinse, bakes piece to be dyed；
9.2 for resin slicer, and the slice, thin piece of 1 μ m-thick for directly cutting ultramicrotome uses specific purpose tool from tank
Inside choose and be placed on the microscope slide of few drops distilled water, 70 DEG C are dried；
（10）Dyeing
Paraffin section conventionally sarranine Fast Green counterstain, the TBO stains that can also use resin slicer to commonly use；Resin slicer
Using staining counter, the slice, thin piece that previous step is glued（Microscope slide）It is placed on roasting piece platform, 40 DEG C, respectively using the process of 1% sarranine
1min, after cleaning, 0.5%TBO processes 1min, and last 0.5% crystal violet is processed, and distilled water bakes piece after cleaning.
Section embedding effect is shown in Fig. 1, Fig. 2.
Embodiment 2
It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud, comprise the following steps：
（1）Sample is drawn materials fixation：Double-edged razor blade extracts the fresh lateral bud rip cutting plucked in each stem section of Caulis Sacchari sinensis, for ending starts the
The bud of a piece of full lamina piece correspondence sections calculates No. 1, is cut downwards according to Research Requirements successively, and sub-bottle is fixed, and per bottle is put into about 2ml
Previous ready 2.5% glutaraldehyde fixative；
（2）Soften：After previous step about 4h, for the sample near top（About 1 ~ No. 4 lateral bud）Select to continue to fix, remaining side
Fixative is extracted out by bud, buffer solution for cleaning three times, per minor tick 10min, is sucked buffer afterwards, is put into 4% ethylenediamine water-soluble
Liquid, stands 10h and softens at 4 DEG C, can 60 min of proper extension set time if close base portion lateral bud is really up to the mark；
（3）Dehydration：By the lateral bud after softening/not through bating step tender shoots in the lump with buffer solution for cleaning three times, every time between
Every 10min, buffer is sucked afterwards, improve 20%, 40%, 60%, 80%, 100% step by step according to alcohol concentration and be dehydrated, often walk
25min, if time of day is too late, can directly switch to 70% ethanol overnight after 60% ethanol, the next day continue.It is thorough to ensure dehydration
Bottom, 100% ethanol need three times, each 20min；
（4）Displacement：
4.1 for the lateral bud for numbering larger close base portion（Harder, volume is larger）, take paraffin section embedding flow process, displacement
Step adopts dimethylbenzene, first removes absolute alcohol, adds dimethylbenzene/ethanol 1:1 mixed liquor, stands 30min, pours out afterwards and use 2 instead:
1 mixed liquor, uses pure dimethylbenzene instead after 30min；
4.2 for the lateral bud of tenderer close ending（It is softer, small volume）, take resin slicer embedding flow process, displacement step
Using expoxy propane, first go in absolute alcohol, sample bottle, to add expoxy propane/ethanol 1:1 mixed liquor, stands 10min, falls afterwards
Go out to use 2 instead:1 mixed liquor, uses pure propylene oxide instead after 10min, a pure propylene oxide is changed per 10min, in triplicate；
（5）Infiltration：
5.1. for paraffin step,, to ooze wax process, the pure dimethylbenzene of previous step adds the paraffin of fusing for this, and temperature is 65 ~
75 DEG C, process time 30min changes the paraffin of fusing, reprocesses 30min, obtains the lateral bud tissue of waxdip
5.2 for resin embedding step, is 1ml by expoxy propane restricted volume in 5.2 steps each sample bottle, then
Deca 0.2ml epoxy resin, mixes, and stands 2h, afterwards Deca 0.2ml resin per hour, improves three mistakes of resin concentration step by step
Afterwards, half mixed liquor is sucked, equal proportion adds second half virgin resin, mixes and stands 3h, changes virgin resin 12h infiltrations, overall process is extremely
Gently shake to promote embedding medium to permeate in low speed shaking table
（6）Embedding：Using the detachable embedding plate of special design；
For 6.1 paraffin sections, using increasing plate；
For 6.2 resin slicers, using basic plate；
（7）Repair block：Save, or only need slightly to grind off a little unnecessary paraffin/resin of lateral bud sample periphery；
（8）Section：
For wax embedding block, base just directly can be cut into slices with the bonding of amonang block, 8 μm of slice thickness；Resin embedding flow process
Sample blocks, its base just can be cut into slices using ultramicrotome on diamant with pedestal resin-bonding, 1 μm of slice thickness
（9）Exhibition piece, bonding die：
Wax band comprising sample after slice, thin piece is cut out gently is provoked using brush pen, is placed on pre- by 9.1 paraffin-embedded samples
First wash and on the microscope slide of Deca few drops distilled water, 50 DEG C of exhibition pieces, roasting piece 30min are brought into close contact microscope slide up to section, with
After insert to stand in pure dimethylbenzene dewaxing cylinder and slough within ten minutes paraffin, tweezers gently take out the microscope slide after dewaxing, are sequentially placed into
100%th, 90%, 70%, 50%, 30%, 10% ethanol enters line replacement, and last microscope slide Deca distilled water carries out rinse, bakes piece to be dyed；
9.2 for resin slicer, and the slice, thin piece of 1 μ m-thick for directly cutting ultramicrotome uses specific purpose tool from tank
Inside choose and be placed on the microscope slide of few drops distilled water, 70 DEG C are dried；
（10）Dyeing
Paraffin section conventionally sarranine Fast Green counterstain, the TBO stains that can also use resin slicer to commonly use；Resin slicer
Using staining counter, the slice, thin piece that previous step is glued（Microscope slide）It is placed on roasting piece platform, 40 DEG C, respectively using the process of 1% sarranine
1min, after cleaning, 0.5%TBO processes 1min, and last 0.5% crystal violet is processed, and distilled water bakes piece after cleaning.
Section embedding effect is shown in Fig. 2, Fig. 3.
Embodiment 3
It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud, comprise the following steps：
（1）Sample is drawn materials fixation：Double-edged razor blade extracts the fresh lateral bud rip cutting plucked in each stem section of Caulis Sacchari sinensis, for ending starts the
The bud of a piece of full lamina piece correspondence sections calculates No. 1, is cut downwards according to Research Requirements successively, and sub-bottle is fixed, and per bottle is put into about 2ml
Previous ready 2.5% glutaraldehyde fixative；
（2）Soften：After previous step about 4h, for the sample near top（About 1 ~ No. 4 lateral bud）Select to continue to fix, remaining side
Fixative is extracted out by bud, buffer solution for cleaning three times, per minor tick 10min, is sucked buffer afterwards, is put into 4% ethylenediamine water-soluble
Liquid, stands 9 h softenings at 4 DEG C, can 45 min of proper extension set time if close base portion lateral bud is really up to the mark；
（3）Dehydration：By the lateral bud after softening/not through bating step tender shoots in the lump with buffer solution for cleaning three times, every time between
Every 10min, buffer is sucked afterwards, improve 20%, 40%, 60%, 80%, 100% step by step according to alcohol concentration and be dehydrated, often walk
22min, if time of day is too late, can directly switch to 70% ethanol overnight after 60% ethanol, the next day continue.It is thorough to ensure dehydration
Bottom, 100% ethanol need three times, each 20min；
（4）Displacement：
4.1 for the lateral bud for numbering larger close base portion（Harder, volume is larger）, take paraffin section embedding flow process, displacement
Step adopts dimethylbenzene, first removes absolute alcohol, adds dimethylbenzene/ethanol 1:1 mixed liquor, stands 30min, pours out afterwards and use 2 instead:
1 mixed liquor, uses pure dimethylbenzene instead after 30min；
4.2 for the lateral bud of tenderer close ending（It is softer, small volume）, take resin slicer embedding flow process, displacement step
Using expoxy propane, first go in absolute alcohol, sample bottle, to add expoxy propane/ethanol 1:1 mixed liquor, stands 10min, falls afterwards
Go out to use 2 instead:1 mixed liquor, uses pure propylene oxide instead after 10min, a pure propylene oxide is changed per 10min, in triplicate；
（5）Infiltration：
5.1. for paraffin step,, to ooze wax process, the pure dimethylbenzene of previous step adds the paraffin of fusing for this, and temperature is 65 ~
75 DEG C, 25 min of process time changes the paraffin of fusing, reprocesses 25 min, obtains the lateral bud tissue of waxdip
5.2 for resin embedding step, is 1ml by expoxy propane restricted volume in 5.2 steps each sample bottle, then
Deca 0.2ml epoxy resin, mixes, and stands 2h, afterwards Deca 0.2ml resin per hour, improves three mistakes of resin concentration step by step
Afterwards, half mixed liquor is sucked, equal proportion adds second half virgin resin, mixes and stands 3h, changes virgin resin 12h infiltrations, overall process is extremely
Gently shake to promote embedding medium to permeate in low speed shaking table
（6）Embedding：Using the detachable embedding plate of special design；
For 6.1 paraffin sections, using increasing plate；
For 6.2 resin slicers, using basic plate；
（7）Repair block：Save, or only need slightly to grind off a little unnecessary paraffin/resin of lateral bud sample periphery；
（8）Section：
For wax embedding block, base just directly can be cut into slices with the bonding of amonang block, 8 μm of slice thickness；Resin embedding flow process
Sample blocks, its base just can be cut into slices using ultramicrotome on diamant with pedestal resin-bonding, 1 μm of slice thickness
（9）Exhibition piece, bonding die：
Wax band comprising sample after slice, thin piece is cut out gently is provoked using brush pen, is placed on pre- by 9.1 paraffin-embedded samples
First wash and on the microscope slide of Deca few drops distilled water, 50 DEG C of exhibition pieces, roasting piece 30min are brought into close contact microscope slide up to section, with
After insert to stand in pure dimethylbenzene dewaxing cylinder and slough within ten minutes paraffin, tweezers gently take out the microscope slide after dewaxing, are sequentially placed into
100%th, 90%, 70%, 50%, 30%, 10% ethanol enters line replacement, and last microscope slide Deca distilled water carries out rinse, bakes piece to be dyed；
9.2 for resin slicer, and the slice, thin piece of 1 μ m-thick for directly cutting ultramicrotome uses specific purpose tool from tank
Inside choose and be placed on the microscope slide of few drops distilled water, 70 DEG C are dried；
（10）Dyeing
Paraffin section conventionally sarranine Fast Green counterstain, the TBO stains that can also use resin slicer to commonly use；Resin slicer
Using staining counter, the slice, thin piece that previous step is glued（Microscope slide）It is placed on roasting piece platform, 40 DEG C, respectively using the process of 1% sarranine
1min, after cleaning, 0.5%TBO processes 1min, and last 0.5% crystal violet is processed, and distilled water bakes piece after cleaning.
Section embedding effect is shown in Fig. 3, Fig. 4.
Embodiment 4
It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud, comprise the following steps：
（1）Sample is drawn materials fixation：Double-edged razor blade extracts the fresh lateral bud rip cutting plucked in each stem section of Caulis Sacchari sinensis, for ending starts the
The bud of a piece of full lamina piece correspondence sections calculates No. 1, is cut downwards according to Research Requirements successively, and sub-bottle is fixed, and per bottle is put into about 2ml
Previous ready 2.5% glutaraldehyde fixative；
（2）Soften：After previous step about 4h, for the sample near top（About 1 ~ No. 4 lateral bud）Select to continue to fix, remaining side
Fixative is extracted out by bud, buffer solution for cleaning three times, per minor tick 10min, is sucked buffer afterwards, is put into 4% ethylenediamine water-soluble
Liquid, stands 8.5 h softenings at 4 DEG C, can 35 min of proper extension set time if close base portion lateral bud is really up to the mark；
（3）Dehydration：By the lateral bud after softening/not through bating step tender shoots in the lump with buffer solution for cleaning three times, every time between
Every 10min, buffer is sucked afterwards, improve 20%, 40%, 60%, 80%, 100% step by step according to alcohol concentration and be dehydrated, often walk
21min, if time of day is too late, can directly switch to 70% ethanol overnight after 60% ethanol, the next day continue.It is thorough to ensure dehydration
Bottom, 100% ethanol need three times, each 20min；
（4）Displacement：
4.1 for the lateral bud for numbering larger close base portion（Harder, volume is larger）, take paraffin section embedding flow process, displacement
Step adopts dimethylbenzene, first removes absolute alcohol, adds dimethylbenzene/ethanol 1:1 mixed liquor, stands 30min, pours out afterwards and use 2 instead:
1 mixed liquor, uses pure dimethylbenzene instead after 30min；
4.2 for the lateral bud of tenderer close ending（It is softer, small volume）, take resin slicer embedding flow process, displacement step
Using expoxy propane, first go in absolute alcohol, sample bottle, to add expoxy propane/ethanol 1:1 mixed liquor, stands 10min, falls afterwards
Go out to use 2 instead:1 mixed liquor, uses pure propylene oxide instead after 10min, a pure propylene oxide is changed per 10min, in triplicate；
（5）Infiltration：
5.1. for paraffin step,, to ooze wax process, the pure dimethylbenzene of previous step adds the paraffin of fusing for this, and temperature is 65 ~
75 DEG C, process time 22min changes the paraffin of fusing, reprocesses 22 min, obtains the lateral bud tissue of waxdip
5.2 for resin embedding step, is 1ml by expoxy propane restricted volume in 5.2 steps each sample bottle, then
Deca 0.2ml epoxy resin, mixes, and stands 2h, afterwards Deca 0.2ml resin per hour, improves three mistakes of resin concentration step by step
Afterwards, half mixed liquor is sucked, equal proportion adds second half virgin resin, mixes and stands 3h, changes virgin resin 12h infiltrations, overall process is extremely
Gently shake to promote embedding medium to permeate in low speed shaking table
（6）Embedding：Using the detachable embedding plate of special design；
For 6.1 paraffin sections, using increasing plate；
For 6.2 resin slicers, using basic plate；
（7）Repair block：Save, or only need slightly to grind off a little unnecessary paraffin/resin of lateral bud sample periphery；
（8）Section：
For wax embedding block, base just directly can be cut into slices with the bonding of amonang block, 8 μm of slice thickness；Resin embedding flow process
Sample blocks, its base just can be cut into slices using ultramicrotome on diamant with pedestal resin-bonding, 1 μm of slice thickness
（9）Exhibition piece, bonding die：
Wax band comprising sample after slice, thin piece is cut out gently is provoked using brush pen, is placed on pre- by 9.1 paraffin-embedded samples
First wash and on the microscope slide of Deca few drops distilled water, 50 DEG C of exhibition pieces, roasting piece 30min are brought into close contact microscope slide up to section, with
After insert to stand in pure dimethylbenzene dewaxing cylinder and slough within ten minutes paraffin, tweezers gently take out the microscope slide after dewaxing, are sequentially placed into
100%th, 90%, 70%, 50%, 30%, 10% ethanol enters line replacement, and last microscope slide Deca distilled water carries out rinse, bakes piece to be dyed；
9.2 for resin slicer, and the slice, thin piece of 1 μ m-thick for directly cutting ultramicrotome uses specific purpose tool from tank
Inside choose and be placed on the microscope slide of few drops distilled water, 70 DEG C are dried；
（10）Dyeing
Paraffin section conventionally sarranine Fast Green counterstain, the TBO stains that can also use resin slicer to commonly use；Resin slicer
Using staining counter, the slice, thin piece that previous step is glued（Microscope slide）It is placed on roasting piece platform, 40 DEG C, respectively using the process of 1% sarranine
1min, after cleaning, 0.5%TBO processes 1min, and last 0.5% crystal violet is processed, and distilled water bakes piece after cleaning.
Embodiment 5
It is a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud, comprise the following steps：
（1）Sample is drawn materials fixation：Double-edged razor blade extracts the fresh lateral bud rip cutting plucked in each stem section of Caulis Sacchari sinensis, for ending starts the
The bud of a piece of full lamina piece correspondence sections calculates No. 1, is cut downwards according to Research Requirements successively, and sub-bottle is fixed, and per bottle is put into about 2ml
Previous ready 2.5% glutaraldehyde fixative；
（2）Soften：After previous step about 4h, for the sample near top（About 1 ~ No. 4 lateral bud）Select to continue to fix, remaining side
Fixative is extracted out by bud, buffer solution for cleaning three times, per minor tick 10min, is sucked buffer afterwards, is put into 4% ethylenediamine water-soluble
Liquid, stands 9.5 h softenings at 4 DEG C, can 50 min of proper extension set time if close base portion lateral bud is really up to the mark；
（3）Dehydration：By the lateral bud after softening/not through bating step tender shoots in the lump with buffer solution for cleaning three times, every time between
Every 10min, buffer is sucked afterwards, improve 20%, 40%, 60%, 80%, 100% step by step according to alcohol concentration and be dehydrated, often walk
23min, if time of day is too late, can directly switch to 70% ethanol overnight after 60% ethanol, the next day continue.It is thorough to ensure dehydration
Bottom, 100% ethanol need three times, each 20min；
（4）Displacement：
4.1 for the lateral bud for numbering larger close base portion（Harder, volume is larger）, take paraffin section embedding flow process, displacement
Step adopts dimethylbenzene, first removes absolute alcohol, adds dimethylbenzene/ethanol 1:1 mixed liquor, stands 30min, pours out afterwards and use 2 instead:
1 mixed liquor, uses pure dimethylbenzene instead after 30min；
4.2 for the lateral bud of tenderer close ending（It is softer, small volume）, take resin slicer embedding flow process, displacement step
Using expoxy propane, first go in absolute alcohol, sample bottle, to add expoxy propane/ethanol 1:1 mixed liquor, stands 10min, falls afterwards
Go out to use 2 instead:1 mixed liquor, uses pure propylene oxide instead after 10min, a pure propylene oxide is changed per 10min, in triplicate；
（5）Infiltration：
5.1. for paraffin step,, to ooze wax process, the pure dimethylbenzene of previous step adds the paraffin of fusing for this, and temperature is 65 ~
75 DEG C, process time 28min changes the paraffin of fusing, reprocesses 28 min, obtains the lateral bud tissue of waxdip
5.2 for resin embedding step, is 1ml by expoxy propane restricted volume in 5.2 steps each sample bottle, then
Deca 0.2ml epoxy resin, mixes, and stands 2h, afterwards Deca 0.2ml resin per hour, improves three mistakes of resin concentration step by step
Afterwards, half mixed liquor is sucked, equal proportion adds second half virgin resin, mixes and stands 3h, changes virgin resin 12h infiltrations, overall process is extremely
Gently shake to promote embedding medium to permeate in low speed shaking table
（6）Embedding：Using the detachable embedding plate of special design；
For 6.1 paraffin sections, using increasing plate；
For 6.2 resin slicers, using basic plate；
（7）Repair block：Save, or only need slightly to grind off a little unnecessary paraffin/resin of lateral bud sample periphery；
（8）Section：
For wax embedding block, base just directly can be cut into slices with the bonding of amonang block, 8 μm of slice thickness；Resin embedding flow process
Sample blocks, its base just can be cut into slices using ultramicrotome on diamant with pedestal resin-bonding, 1 μm of slice thickness
（9）Exhibition piece, bonding die：
Wax band comprising sample after slice, thin piece is cut out gently is provoked using brush pen, is placed on pre- by 9.1 paraffin-embedded samples
First wash and on the microscope slide of Deca few drops distilled water, 50 DEG C of exhibition pieces, roasting piece 30min are brought into close contact microscope slide up to section, with
After insert to stand in pure dimethylbenzene dewaxing cylinder and slough within ten minutes paraffin, tweezers gently take out the microscope slide after dewaxing, are sequentially placed into
100%th, 90%, 70%, 50%, 30%, 10% ethanol enters line replacement, and last microscope slide Deca distilled water carries out rinse, bakes piece to be dyed；
9.2 for resin slicer, and the slice, thin piece of 1 μ m-thick for directly cutting ultramicrotome uses specific purpose tool from tank
Inside choose and be placed on the microscope slide of few drops distilled water, 70 DEG C are dried；
（10）Dyeing
Paraffin section conventionally sarranine Fast Green counterstain, the TBO stains that can also use resin slicer to commonly use；Resin slicer
Using staining counter, the slice, thin piece that previous step is glued（Microscope slide）It is placed on roasting piece platform, 40 DEG C, respectively using the process of 1% sarranine
1min, after cleaning, 0.5%TBO processes 1min, and last 0.5% crystal violet is processed, and distilled water bakes piece after cleaning.
Fig. 7 be middle part lateral bud paraffin section rip cutting figure, 5 × object lens；It can be seen that section is substantially complete, there is no breakage；Fig. 8 is
Prior paraffin Sectioning（Without bating step）The piece subgraph made；5 × object lens amplify, and are difficult to cut out complete section, have breakage；
Fig. 9 be bottom lateral bud rip cutting, 5 × object lens；Figure 10 be paraffin section figure, the most hard lateral bud of base portion（15+ sections）, 5 × object lens put
Big multiple；It can be seen that rip cutting section is complete, perula cell is intact, no serious damage, it was demonstrated that processing method/flow process reaches
Expected effect.
Claims (3)
- It is 1. a kind of to embed flaking method for Caulis Sacchari sinensis Different node lateral bud, it is characterised in that：Comprise the following steps：（1）Sample is drawn materials fixation：Double-edged razor blade extracts the fresh lateral bud rip cutting plucked in each stem section of Caulis Sacchari sinensis, for ending starts the The bud of a piece of full lamina piece correspondence sections calculates No. 1, is cut downwards according to Research Requirements successively, and sub-bottle is fixed, and per bottle is put into about 2ml Previous ready 2.5% glutaraldehyde fixative；（2）Soften：After previous step about 4h, for the sample near top selects to continue to fix, fixative is taken out by remaining lateral bud Go out, buffer solution for cleaning three times, per minor tick 10min, suck buffer afterwards, be put into 4% ethylenediamine solution, stand 8 at 4 DEG C ~ 10h softens, can 30 ~ 60 min of proper extension set time if close base portion lateral bud is really up to the mark；（3）Dehydration：By the lateral bud after softening/not through bating step tender shoots in the lump with buffer solution for cleaning three times, every time between Every 10min, buffer is sucked afterwards, improve 20%, 40%, 60%, 80%, 100% step by step according to alcohol concentration and be dehydrated, often walk 20 ~ 25min, if time of day is too late, can directly switch to 70% ethanol overnight after 60% ethanol, the next day continue, be ensure dehydration Thoroughly, 100% ethanol needs three times, each 20min；（4）Displacement：A. for the lateral bud for numbering larger close base portion, paraffin section embedding flow process, displacement step is taken to adopt dimethylbenzene, Absolute alcohol is first removed, dimethylbenzene/ethanol 1 is added:1 mixed liquor, stands 30min, pours out afterwards and use 2 instead:1 mixed liquor, after 30min Use pure dimethylbenzene instead；B. for the lateral bud of tenderer close ending, resin slicer embedding flow process, displacement step is taken to adopt expoxy propane, first Go in absolute alcohol, sample bottle, to add expoxy propane/ethanol 1:1 mixed liquor, stands 10min, pours out afterwards and use 2 instead:1 mixing Liquid, uses pure propylene oxide instead after 10min, a pure propylene oxide is changed per 10min, in triplicate；（5）Infiltration：A. for paraffin step,, to ooze wax process, the pure dimethylbenzene of previous step adds the paraffin of fusing for this, and temperature is 65 ~ 75 DEG C, 20 ~ 30min of process time changes the paraffin of fusing, reprocesses 20 ~ 30min, obtains the lateral bud tissue of waxdip；B., for resin embedding step, it is 1ml by expoxy propane restricted volume in 5.2 steps each sample bottle, then Deca 0.2ml epoxy resin, mixes, and stands 2h, afterwards Deca 0.2ml resin per hour, improves three mistakes of resin concentration step by step Afterwards, half mixed liquor is sucked, equal proportion adds second half virgin resin, mixes and stands 3h, changes virgin resin 12h infiltrations, overall process is extremely Gently shake to promote embedding medium to permeate in low speed shaking table；（6）Embedding：Using the detachable embedding plate of special design；A. for paraffin section, using increasing plate；B. for resin slicer, using basic plate；（7）Repair block：Save, or only need slightly to grind off a little unnecessary paraffin/resin of lateral bud sample periphery；（8）Section：For wax embedding block, base just directly can be cut into slices with the bonding of amonang block, 8 μm of slice thickness；Resin embedding flow process Sample blocks, its base just can be cut into slices using ultramicrotome on diamant with pedestal resin-bonding, 1 μm of slice thickness；（9）Exhibition piece, bonding die：A. the wax band comprising sample is gently provoked after slice, thin piece is cut out by paraffin-embedded sample using brush pen, is placed in advance On the microscope slide of washed and Deca few drops distilled water, 50 DEG C of exhibition pieces, roasting piece 30min are brought into close contact microscope slide up to section, subsequently Insert to stand in pure dimethylbenzene dewaxing cylinder and slough within ten minutes paraffin, tweezers gently take out the microscope slide after dewaxing, are sequentially placed into 100%th, 90%, 70%, 50%, 30%, 10% ethanol enters line replacement, and last microscope slide Deca distilled water carries out rinse, bakes piece to be dyed；B., for resin slicer, the slice, thin piece of 1 μ m-thick for directly cutting ultramicrotome is using specific purpose tool from tank Choose and be placed on the microscope slide of few drops distilled water, 70 DEG C are dried；（10）DyeingParaffin section conventionally sarranine Fast Green counterstain, the TBO stains that can also use resin slicer to commonly use；Resin slicer Using staining counter, the microscope slide that previous step is glued is placed on roasting piece platform, 40 DEG C, 1min is processed using 1% sarranine respectively, clean 0.5%TBO processes 1min afterwards, and last 0.5% crystal violet is processed, and distilled water bakes piece after cleaning.
- 2. according to claim 1 a kind of for Caulis Sacchari sinensis Different node lateral bud embedding flaking method, it is characterised in that：Step （1）2.5% described glutaraldehyde fixative, is buffered with 0.05mol/L pH7.2 phosphate buffers or 0.05mol/L cacodylic acids Liquid is prepared, and latter of which is better, and adds 1% sucrose, 4 DEG C of preservations after preparation.
- 3. according to claim 1 a kind of for Caulis Sacchari sinensis Different node lateral bud embedding flaking method, it is characterised in that：Step （5）Described epoxy resin, its formula is resin embedding host, the mass ratio of plasticiser, sclerosing agent, catalyst amount is 10: 5.5:26:0.4。
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| CN114689407A (en) * | 2022-04-14 | 2022-07-01 | 四川大学华西医院 | Method for manufacturing paraffin section of animal microtissue |
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| CN112903406B (en) * | 2021-03-25 | 2023-03-10 | 中南林业科技大学 | Method for making paraffin section of tree thin root |
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