CN110361239A - A kind of method of nerve fibre bundle tracer in insect light color feeler - Google Patents
A kind of method of nerve fibre bundle tracer in insect light color feeler Download PDFInfo
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- CN110361239A CN110361239A CN201910624985.8A CN201910624985A CN110361239A CN 110361239 A CN110361239 A CN 110361239A CN 201910624985 A CN201910624985 A CN 201910624985A CN 110361239 A CN110361239 A CN 110361239A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The invention discloses a kind of methods of nerve fibre bundle tracer in insect light color feeler, belong to laboratory sample processing technology field.The present invention is fixed by living body feeler nickel chloride or cobalt chloride dip dyeing, living body feeler and micro- separation, colour developing, fixation, dehydration, feeler and the brain transparency process of brain, feeler posture and the tracer of nerve fibre bundle in complete feeler can be realized in photomicrograph.The present invention solve the problems, such as be not sliced do not destroy feeler under conditions of complete feeler is transparent and its nerve fibre bundle multi-angle visible, also allow for directly observing coupling for nerve fibre bundle and brain in feeler in nerve pathway level；The method of the present invention is low in cost, method is reliable, easy to operation, reproducible, can directly obtain the data of the various dimensions of feeler intrinsic nerve structure.
Description
Technical field
The present invention relates to a kind of methods of nerve fibre bundle tracer in insect light color feeler, belong to laboratory sample processing technique
Field.
Background technique
Feeler is most important for the existence and procreation of insect.Mainly pass through feeler experiences smell, wind, temperature and humidity to insect
Etc. external environments variation.By means of feeler, insect can carry out search of food or spouse, hide natural enemy, plant in communication and
Find a series of vital movement such as suitable spawning habitat.According to morphological differences, the feeler of insect can be divided into setiform, tool awns
The types such as shape, filiform, club-like.Feeler is divided into three section of scapus, pedicel and flagellum, and surface is covered with the outer of one layer of chitin
Bone has protection soft inner structure and reduces the functions such as moisture evaporation, and different colours are presented in the feeler of variety classes insect,
Such as the black feeler of Apidae, the light feeler of Tephritidae, the light feeler of Drosophilidae.
Feeler is a multi-modal sense organ.Smell, wind feel, sound sensation, temperature sensation, humidity are dispersed on feeler surface
The receptor of the different modalities such as feel, for example hair sensilla, sensillum basiconicum, stick sense are mainly dispersed on the feeler of dipteral insect
The sensors such as device, chamber sensillum basiconicum, arista, sensory pit, further functional study show, insect hair sensilla, taper sense
Device and wooden club shape sensor, surface are distributed " close hole ", and function is detection smell.Scent molecule enters through duct, with nerve cell
Odorant receptor is converted into electricity physiological signal after combining on film, along nerve fibers conduct to brain.Chamber sensillum basiconicum may be impressionization
Or temperature and humidity；Arista can experience temperature；Arista and flagellum may be conducted mechanical signal to stalk by its displacement
Section is internal, experiences wind, gravity and sound etc. by the Jiang Shi device nerve cell inside pedicel；Sensory pit is likely to experience on flagellum
Humidity.The different types of stimulation such as extraneous chemical odor, sound, temperature and humidity is converted electricity physiological signal by these sensors, and
It is transferred to intracerebral by the Sensory nerve fibre beam of different modalities corresponding inside feeler, the antennal lobe and feeler machine with intracerebral
The formation such as tool sensorimotor maincenter closely couple.These are fine as the long range sensory nerve of terminal using brain using feeler as starting point
Beam is tieed up, is nerve of the insect realization to surrounding enviroment and target apperception, the neural basal of navigator fix and pest green prevention and control
Target spot.Therefore the nerve fibre bundle of research feeler is most important, but so far, people are to insect feeler and its primary feeling
The Nervous System Anatomy of nerve pathway (nerve fibre bundle) is substantially unclear.
Carry out the feeler anatomy of insect feeler such as model insects Drosophilidae Drosophila melanogaster using the microtomies such as crosscutting, longitudinal sectional
Research largely destroys the structure of nerve fibre bundle in feeler, therefore the information of the antennae nerve fibre bundle obtained is
Fragmentation, accuracy are lower, and it is intuitive cannot accurately to obtain the true quantity of nerve fibre bundle in feeler, spatial distribution and structure etc.
Information.Also, microtomy destroys coupling for feeler and brain, can not be observed in an intact nervous access level.
Therefore, the full feeler primary sensory nerve access of insect cannot be parsed in situ so far in the world, including the nerve tract in feeler
The information such as quantity, spatial distribution and structure.
In the prior art, two kinds of Neural tracers of nickel chloride and cobalt chloride are injected Drosophila melanogaster feeler by Stocker et al.
After the different parts (being dispersed with different type sensor) of flagellum, the sensor of discovery flagellum different parts can project intracerebral feeler
The different target areas of leaf.Then, both Neural tracers in other different types of insect such as moth class, cricket similarly by with
In research of the feeler sensory nerve in intracerebral projection target area；Lysine cobalt is observed Agrotis by Xiang Hui and Peng Bo
As a result the olfactory nerves projection tract of segetum hero moth only obtains antennae nerve in the projection situation of intracerebral.
This existing two kinds of international and domestic researchs of Neural tracer, are only limitted to brain, and nerve fibre bundle is studied in the feeler of periphery
It is not reported so far, the primary of antennae nerve fibre bundle to brain feels that access research is not reported so far.Main cause be to
Obtain the information of accurate antennae nerve fibre bundle, it is necessary to directly be observed on intact feeler, it is necessary in multiple steps
On overcome important technical challenge, for example, dip dyeing step control, color developing agent rate-determining steps, the discovery of feeler clarifier and application,
Feeler posture fixation etc..The solution of these problems will bring far-reaching shadow to the Nervous System Anatomy of insect feeler sensory nerve access
It rings.
The feeler of different insects is of different sizes, and shape is different, and color is different, even if can be marked with Neural tracer
Note, but tracer dip dyeing duration is not easy to control, too long it will cause tracers in the non-specific diffusion of nervous system, it is too short then
Nerve fibre bundle cannot be all labeled, and these can all directly affect the color developing effect in later period.In process color, it is difficult to control
Develop the color duration, be easy to cause excessive color development.These reasons cause nerve fibre bundle in feeler to be difficult to exclusively be marked.In addition,
The feeler of insect is wrapped up by the ectoskeleton of one layer of chitin, not only seriously affects colour developing efficiency and process, but also even if nerve
Fibre bundle is labeled to be come out, and lighttight cuticula, which results in, can not carry out optical observation.Therefore, nerve fibre in complete feeler
The research of beam it is sluggish always not before.Meanwhile in order to study Antennal Sensilla official comprehensively, need to feeler external and internal compositions and function into
The observation and inspection of row various dimensions.The feeler size of many insects is smaller, such as 860-1000 μm of overall length of Bactrocera correcta feeler, most
It is about 170 μm wide, about 150 μm of thickness, and it is divided into scapus, pedicel and flagellum again, and respectively save form, structure snd size difference, it is whole
Irregular geometry is presented in body, and the fulcrum needed when fixed, by natural placement, can not achieve multidimensional on different curve
The posture of degree is fixed.Several fixing means to pass through in the world at present mainly have paraffin embedding or Agarose embedding and glass slide solid
It is fixed.Paraffin or Agarose embedding technique for fixing can not achieve the sight to complete feeler surface or internal structure under an optical microscope
It examines.Under normal circumstances, cell or tissue slice (incomplete organ) is usually placed on glass slide by production observation sample in the world,
Coverslip is pressed in thereon, is used as observation after mounting for a long time.Need histotomy smooth in favor of coverslip pressing.Once
This processing is done, the posture of slice cannot be adjusted again, need not be also adjusted, and the image of acquisition is in a dimension
's.The stitching image obtained by tactile organ slice, heavy workload, the distortion factor is high, therefore, under conditions of not being sliced directly
Obtaining tactile organ image of internal structure information is the target that international scientific circle has been working hard pursuit.However, feeler is one
The organ of form irregular can not achieve inside, outside, tiltedly outside, tiltedly inside, back with tabletting method on traditional glass slide
The posture of the special angles such as side, veutro is fixed, optical observation and image obtain.
Summary of the invention
Aiming at the problem that being difficult to carry out nerve fibre bundle tracer in whole insect light color feeler in the prior art, the present invention is mentioned
The method for having supplied nerve fibre bundle tracer in a kind of insect light color feeler, the present invention are soaked by living body feeler nickel chloride or cobalt chloride
Dye, living body feeler and micro- separation, colour developing, fixation, dehydration, feeler and the brain transparency process of brain, feeler posture fix, are micro-
It takes pictures and the tracer of nerve fibre bundle in complete feeler can be realized；The limitation for breaching existing microtomy establishes light touching
The transparent technology at angle realizes nerve fibre bundle visualized in situ in feeler, obtain nerve fibre bundle quantity in complete feeler,
The fine informations such as spatial distribution and travelling route.
A kind of method of nerve fibre bundle tracer in insect light color feeler, the specific steps are as follows:
(1) living insects feeler carries out nickel chloride solution or cobalt chloride solution dip dyeing；
(2) micro- separation living insects feeler and brain；
(3) labeled to insect feeler and brain epineural fibre bundle using rubeane acid solution submergence insect feeler and brain, it inhales
Except rubeane acid solution, then using alcohol washes developed the color after insect feeler and brain；
(4) successively using the tissue in the insect feeler and brain of the fixed colour developing of formalin A and formalin B, then to elder brother
Worm feeler and brain are carried out dehydrating；
(5) insect feeler and brain are immersed in gaultherolin, handles 1~12h at room temperature；
(6) posture of insect feeler and brain is adjusted and fixed, then carries out photomicrograph and obtains different angle, different times magnifications
The image information of nerve fibre bundle in rate inferior antenna.
The mass concentration of nickel chloride solution or cobalt chloride solution is 1%~10% in the step (1), if insect feeler is long
Degree is n millimeters, and immerged time is n~n+5 hours.
Alcohol is absolute alcohol in the step (3).
The mass concentration of step (4) the formalin A is 5%, and the mass concentration of formalin B is 10%, and formaldehyde is molten
The liquid A set time is 10min, and the formalin B set time is 10min.
The specific method of step (4) dehydration is successively to use mass concentration for 70%, 80%, 90%, 100%
Alcohol insect feeler and insect brain are carried out dehydrating, the dehydration of alcohol time of various concentration is 5min.
The posture of insect feeler and insect brain is fixed in the step (6) uses The Small Well device, and stone is arranged in The Small Well device
Sand.
Further, the quartz sand is two different sizes；The partial size of quartz sand is the 1/5- of sample minimum dimension
1/2, the mass ratio of two kinds of partial size quartz sand is 1:(1~3).There can be more groups using the quartz sand of different sizes and ratio
It closes, produces the fulcrum curved surface of more different heights, that is, can manufacture using two kinds of smaller size of quartz sand combinations
Any angle of feeler posture is fixed in the fulcrum curved surface of different height, realization.The undersized of quartz sand is unfavorable for operating, mistake
It is big to be unfavorable for producing the curved surface for supporting small feeler again.
Preferably, step (1) living insects feeler carries out the concrete operations side of nickel chloride solution or cobalt chloride solution dip dyeing
Method is
According to the size of insect, the sample-adding pipette tips of suitable micro sample adding appliance are selected, pipette tips tail end is cut, then by insect
Pipette tips tail end is put into after frost anesthesia, and by micro- power to pushing at pipette tips tip, so that insect head is consolidated at pipette tips tip
It is fixed, carefully expose feeler；Meanwhile absorbent cotton is filled at pipette tips tail end, and pushed forward with thin tweezer, thus fixed insect, rifle
Head is fixed in the plasticine or dentistry wax on glass slide；It is carefully cut at feeler flagellum tip under anatomical lens with eye scissors
A notch out；Meanwhile another end position on same glass slide, the glass capillary containing nickel chloride or cobalt chloride solution
Or glass electrode is fixed in another piece of plasticine or dentistry wax, by the glass capillary containing nickel chloride or cobalt chloride solution
Or glass electrode entangles feeler incision, is immersed in incision in nickel chloride or cobalt chloride solution, keeps clear；Then will
Glass slide immigration is placed in the glass culture dish of wet filter paper item, keeps 50%~70% humidity, and it is small to place 2~48 at room temperature
When.
Further, the tip aperture of glass capillary or glass electrode is wide 1-2.5 times of insect feeler.
Further, the volume for being loaded pipette tips is 10 μ L, 200 μ L or 1000 μ L.
Preferably, the concrete operation method of the micro- separation living insects feeler of step (2) and brain is
Ringer liquid is embedded in cooling in ice and obtains ice-cold Ringer liquid, is persistently filled with 95% in ice-cold Ringer liquid
The mixed gas of oxygen and 5% carbon dioxide；Ice-cold Ringer liquid after being inflated in drop on glass slide, it is then fast with eye scissors
The insect head of the fast related feeler of clip, and put it into the Ringer liquid on glass slide, under Stereo microscope, use tip
From the beginning tweezers and eye scissors are promptly completely separated the brain with feeler in capsule, and removed with tweezers in 3~5 minutes
Tracheae, the air bag on decerebrate surface, and use the sharp triangular tip of syringe needle by the pedicel cutin with flagellum junction
Layer part is cut off, and then, is cleaned 3 times using Ringer liquid.
Preferably, step (3) colour developing uses observes under Stereo microscope.
Preferably, step (4) tissue is fixed uses PCR pipe, and dehydration uses PCR pipe.
Step (5) insect feeler and brain immerse feeler ectoskeleton cuticula, feeler inner tissue and brain in gaultherolin
It is transparent.
Preferably, the concrete operation method of the posture of step (6) adjustment and fixed insect feeler and insect brain is
By the insect feeler handled well and brain metastes on the quartz sand into the The Small Well device of the fixed device of posture, and small
Instilled in well device it is low sticky degree high-transmittance can persistence feeler liquid chemical, carefully moved with tip tweezers
Feeler and quartz sand as needed can carry out feeler from the different directions such as back side, veutro, outside, inside and top and angle
Fixed and pose adjustment.
Further, the height of The Small Well device be no more than 5mm, it is low sticky degree high-transmittance can persistence feeler liquid
State chemical substance is gaultherolin；The excessive height of The Small Well device leads to contain quartz sand and particular liquid chemical substance
Thickness increases, and reduces translucency, is unfavorable for the observation of feeler internal structure, while The Small Well device is excessively high that will to be not easy to posture micro-
The observation reconciled under high power objective, to also influence the close-up of feeler internal structure.
Nerve fibre bundle tracing exploration in insect light color feeler of the present invention:
Using nickel chloride or cobalt chloride as Neural tracer, nickel ion or cobalt are controlled by control tracer immerged time
The distance that ion enters the quantity of nerve and transmits backward guarantees that the activity of insect facilitates the special of tracer during dip dyeing
The dip dyeing of one property, i.e., nickel ion or cobalt ions are travelled rearwardly only along aixs cylinder (nerve fibre bundle), and can be deposited in the distance of traveling
In, as one of the committed step of tracer decides the result of colour developing.The humidity that 50%-70% is provided during dip dyeing can
To reduce the evaporation of tracer aqueous solution, conducive to the osmotic pressure for maintaining tracer aqueous solution, reactor solution is reduced to nerve cell
Influence；During microdissection, Ringer liquid is carried out being filled with mixed gas and lower temperature is kept to touch also for guarantee
The excellent activity of the tissue and cell of angle and brain, tracer is diffused into other non-neural groups when preventing cell activity from reducing or is dead
In knitting, subsequent color developing effect is influenced；Rubeanic acid can form darkviolet complex compound with nickel ion or cobalt ions, thus by nickel from
Darkviolet is dyed in the place of son or cobalt ions accumulation, and then the nerve fibre bundle inside feeler is dyed darkviolet, in colour developing
The duration for controlling colour developing in the process is most important, stops colour developing immediately after nerve fibre bundle is coloured in feeler, removes rubeane
Acid solution, and with alcohol washes, prevent excessive color development, as one of the committed step of tracer effect；Respectively with 5% and 10%
Formalin to after colour developing feeler and brain carry out tissue and fix；Again with 70%, 80%, 90% and 100% graded ethanol into
Row dehydration；Gaultherolin is a kind of colorless and transparent liquid, and refractive index with higher is normally used for the saturating of soft tissue
It is bright；But since insect feeler cuticula is thicker, translucency is low, hinders the visualization of feeler internal structure.
It is found by many experiments, gaultherolin is capable of the cuticula ectoskeleton of transparent feeler light color to a certain extent.
Therefore, insect light color feeler ectoskeleton cuticula, feeler inner tissue and capsules of brain transparence are realized using gaultherolin；Opposition
The feeler or brain of body structure carry out various dimensions pose adjustment and fixation, and as depth obtains nerve fibre bundle image information in feeler
Key link, utilize the stone of solid irregular, translucency, different sizes than insect feeler smaller szie quartz sand
Sand ratio can combination manufacture different height fulcrum curved surface the characteristics of, realize to feeler posture it is any adjustment with fixation, together
When, it is low sticky to spend the unobstructed using the Auxiliary support, the optical path that play the fixation of feeler posture of high transparency gaultherolin and touch
The triple role of angle preservation thoroughly solves the problems, such as fixation, the permanent preservation of the small feeler of insect, has thoroughly removed inside feeler
The last obstacle of structures visualization link.
Beneficial effects of the present invention:
(1) present invention breaches the limitation of existing microtomy, establishes the transparent technology of light feeler, realizes feeler
It is smart to obtain nerve fibre bundle quantity, spatial distribution and travelling route etc. in complete feeler for interior nerve fibre bundle visualized in situ
Thin information brings far-reaching influence to insect antennae nerve anatomy and functional study；
(2) present invention passes through the separation of living body feeler and brain association, and overcoming over can only be fine to insect antennae nerve
The intracerebral of dimension beam projects the limitation analyzed and observed, and directly realizes in primary sensory nerve access level in feeler
The visualization of nerve fibre bundle and brain coupled；
(3) present invention is passed through using the fixed device of feeler utilizes quartz sand more smaller size of than feeler and 2 kinds of quartz sand
Combination dexterously realizes the fixation to same feeler posture and carries out the optical observation of the feeler brain internal structure of various dimensions,
It is easy to operate；
(4) present invention is using the fixed device of feeler, and after the quartz sand of selection suitable dimension, it is three-dimensional to can be applied to other tools
The fixation and observation (such as insect brain or foot) of the tiny organism sample posture of structure, acquisition sample outer surface or internal structure
Various dimensions information；
(5) present invention is low in cost, method is reliable, easy to operation, reproducible.
Detailed description of the invention
Fig. 1 is Technology Roadmap of the invention；
Fig. 2 is insect feeler posture various dimensions fixing device schematic diagram of the present invention, wherein 1- The Small Well device, 2- quartz sand；
Fig. 3 is the visualization figure of the Bactrocera correcta antennae nerve fibre bundle that the present invention obtains and its intracerebral projection, arrow
Indicate nerve fibre bundle,
Fig. 4 is the structure chart of the nerve fibre bundle for the Bactrocera correcta feeler flagellum all angles that the present invention obtains, Fig. 4 A
It is from feeler outside with 4B, from Fig. 4 C is on the inside of feeler, for Fig. 4 D from feeler back side, arrow indicates nerve in feeler
Fibre bundle.
Specific embodiment
Invention is further described in detail With reference to embodiment, but protection scope of the present invention and unlimited
In the content.
Embodiment 1: the feeler of Bactrocera correcta is divided into scapus, pedicel and flagellum, overall length about 860-1000 μ in tool aristiform
M, most about 170 μm wide, most about 100 μm narrow, it is that a size is smaller on the whole, in irregular that each shape and size that save are different
Geometry, and gathered the sense organ of the multiple functions such as smell, the sense of hearing, temperature and humidity feeling and gravity feeling；
A kind of method of nerve fibre bundle tracer in Bactrocera correcta feeler (see Fig. 1), the specific steps are as follows:
(1) feeler nickel chloride is disseminated: according to the size of Bactrocera correcta, selecting the sample loading gun of the micro sample adding appliance of 200 μ L
Head, and cut pipette tips tail end, then will be put into pipette tips tail end after Bactrocera correcta frost anesthesia, and by micro- power to pipette tips tip
Place is pushed, so that insect head is fixed at pipette tips tip, exposes feeler, pipette tips are fixed on plasticine or tooth on glass slide
In section's wax；A notch is carefully cut at feeler flagellum tip with eye scissors under anatomical lens；Meanwhile on same glass slide
Another end position, the glass capillary containing mass concentration for 3% nickel chloride aqueous solution are fixed on another piece of plasticine or tooth
In section's wax, the glass capillary containing nickel chloride aqueous solution is carefully entangled into feeler incision, makes incision submergence in the solution,
It keeps clear；Then glass slide immigration is placed in the glass culture dish of wet filter paper item, keeps 50%~70% humidity, room temperature
It is lower to place 2 hours；
(2) living body feeler and the micro- separation of brain: the Ringer liquid of 50mL is poured into 100mL beaker, and beaker is embedded in
Cooling obtains ice-cold Ringer liquid in ice, and the mixed of 95% oxygen and 5% carbon dioxide is persistently filled in ice-cold Ringer liquid
Close gas；Ice-cold Ringer liquid after being inflated in drop on a glass slide, then with the related feeler of the quick clip of eye scissors
The head of Bactrocera correcta, and put it into the Ringer liquid on glass slide, under Stereo microscope, use tip tweezers and eye
Section cuts, and in 3~5 minutes, promptly completely from the beginning separates the brain with feeler in capsule, and remove decerebrate surface with tweezers
Tracheae, air bag, and using the sharp triangular tip of 1mL syringe needle by the pedicel cuticula part with flagellum junction
Then excision is cleaned 3 times using Ringer liquid；
(3) it develops the color: dripping upper 2 drop rubeane acid solution on clean feeler and brain, observation is aobvious at any time under stereomicroscope
Pornographic condition；Once feeler and intracerebral nerve tract are labeled out, then rubeane acid solution quickly are absorbed with dry filter paper item, be used in combination
100% alcohol washes 3 times remove rubeanic acid extra inside and outside tissue, prevent excessive color development；
(4) fixed: lightly the tissue after colour developing to be transferred in a clean PCR pipe with tip tweezers, successively in drop
The formalin that 2-4 drop mass concentration is 5% and mass concentration is 10%, the formalin of various concentration fix 10min respectively,
Formalin is absorbed with dry filter paper item after fixing every time；
(5) be dehydrated: with tip tweezers lightly by after fixation Bactrocera correcta feeler and brain metastes to another completely
PCR pipe in, successively with mass concentration be 70%, 80%, 90%, 100% alcohol be carried out dehydrating, the wine of various concentration
Essence is each respectively to be dehydrated 5 minutes；
(6) transparency of organization: by the Bactrocera correcta feeler and brain immersion gaultherolin after fixation, room temperature condition is transferred
1~12h is set, keeps feeler ectoskeleton cuticula, feeler inner tissue and brain transparent；
(7) feeler posture is fixed: under Stereo microscope, the insect feeler handled well and brain metastes being filled to posture is fixed
On the quartz sand in The Small Well device set, and instill in The Small Well device low sticky degree high-transmittance can persistence feeler
Liquid chemical carefully moves feeler and quartz sand with tip tweezers, as needed, can be by feeler from back side, veutro, outer
The different directions such as side, inside and top and angle are fixed and pose adjustment；
For the fixed device of posture as shown in Fig. 2, quartz sand 2 is arranged in The Small Well device 1, the partial size of quartz sand is 25 μm (i.e. 500
Mesh) and 38 μm (i.e. 400 mesh), the mass ratio of 500 mesh quartz sands and 400 mesh quartz sands is 1:2；
(8) photomicrograph: feeler and brain after posture is fixed are placed under microscope and obtain different enlargement ratio inferior antennas
The image information of interior nerve fibre bundle adjusts the posture of feeler and fixation as needed, and the image information of angle needed for obtaining is shown in
Fig. 3 and Fig. 4；Wherein, arrow meaning is nerve fibre bundle and its to project the path of intracerebral in feeler in Fig. 3；It is white in Fig. 4
Arrow meaning is nerve fibre bundle in labeled flagellum, and A and B are the number of the nerve fibre bundle in feeler flagellum from outside
Amount, structure and distribution map, C are quantity, structure and the distribution map that nerve fibre bundle in feeler flagellum is observed in inside, and D is back side sight
Examine feeler flagellum nerve fibre bundle quantity, structure and distribution map.
Embodiment 2: citrus fruit fly feeler is divided into scapus, pedicel and flagellum, and about 1030 μm of overall length, wherein scapus is about 190 μ
M, about 120-160 μm wide, pedicel is about 230 μm, and about 90-170 μm wide, pedicel is about 630 μm, and wide about 150-178, middle part is slightly
Thickness, the widest part is about 180 μm of flagellum base portion, most narrow to be in about 90 μm of pedicel base portion, and each size and shape that save are different, whole to present
Irregular geometry；
A kind of method of nerve fibre bundle tracer in citrus fruit fly feeler, the specific steps are as follows:
(1) feeler cobalt chloride is disseminated: according to the size of citrus fruit fly, the sample-adding pipette tips of the micro sample adding appliance of 200 μ L are selected,
And pipette tips tail end is cut, it then will be put into pipette tips tail end after citrus fruit fly frost anesthesia, and by micro- power to pushing away at pipette tips tip
It squeezes, so that insect head is fixed at pipette tips tip, exposes feeler, pipette tips are fixed on plasticine or dentistry wax on glass slide
In；A notch is carefully cut at feeler flagellum tip with eye scissors under anatomical lens；Meanwhile it is another on same glass slide
End position is fixed in another piece of plasticine or dentistry wax containing the glass electrode that mass concentration is 6% cobalt chloride solution, small
Glass electrode containing nickel chloride aqueous solution is entangled feeler incision by the heart, is made incision submergence in the solution, is kept clear；So
Glass slide immigration is placed in the glass culture dish of wet filter paper item afterwards, keeps 50%-70% humidity, it is small to place 2.5 at room temperature
When；
(2) the micro- removing of feeler and brain: the Ringer liquid of 50mL is poured into 100mL beaker, and beaker is embedded in ice
Middle cooling obtains ice-cold Ringer liquid, and the mixed of 95% oxygen and 5% carbon dioxide is persistently filled in ice-cold Ringer liquid
Gas is closed, the Ringer liquid after inflating in drop on glass slide；The head that citrus fruit fly is quickly cut with eye scissors, puts it into load
In Ringer liquid on slide, promptly from the beginning the brain with feeler is completely separated in capsule using tip tweezers and eye scissors
Come, guarantees that feeler feels the complete structure of access, and remove tracheae, the air bag on decerebrate surface.With the sharp triangle of 1ml syringe needle
Shape tip will cover the pedicel stratum corneum ablation in part outstanding on flagellum base portion, to help the colour developing of the micro- beam of nerve in feeler；
Feeler and brain are cleaned 3 times with Ringer liquid again, whole process is completed in 3-5min；
(3) it develops the color: dripping upper 2-3 drop rubeane acid solution on clean feeler and brain, observed at any time under stereomicroscope
Develop the color situation；Once feeler and intracerebral nerve tract are labeled out, then rubeane acid solution quickly are absorbed with dry filter paper item, be used in combination
100% alcohol washes 3 times remove extra rubeanic acid inside and outside tissue, prevent excessive color development；
(4) fixed: lightly the tissue after colour developing to be transferred in a clean PCR pipe with tip tweezers, successively in drop
The formalin that 2-4 drop mass concentration is 5% and 10%, the formalin of various concentration fixes 10min respectively, after fixing every time
Formalin is absorbed with dry filter paper item；
(5) be dehydrated: with tip tweezers lightly by after fixation citrus fruit fly feeler and brain metastes to another completely
It in PCR pipe, is successively dehydrated with the alcohol that mass concentration is 70%, 80%, 90%, 100%, the alcohol difference of various concentration
Dehydration 5 minutes；
(6) it transparency of organization: by the citrus fruit fly feeler and brain immersion gaultherolin after fixation, places under room temperature
1-12h keeps feeler ectoskeleton cuticula, feeler inner tissue and brain transparent.
(7) feeler posture is fixed: under Stereo microscope, the insect feeler handled well and brain metastes being filled to posture is fixed
On the quartz sand in The Small Well device set, and instill in The Small Well device low sticky degree high-transmittance can persistence feeler
Liquid chemical carefully moves feeler and quartz sand with tip tweezers, as needed, can be by feeler from back side, veutro, outer
The different directions such as side, inside and top and angle are fixed and pose adjustment；
For the fixed device of posture as shown in Fig. 2, quartz sand 2 is arranged in The Small Well device 1, the partial size of quartz sand is 500 mesh and 400
The mass ratio of mesh, 500 mesh quartz sands and 400 mesh quartz sands is 1:2；
(8) photomicrograph: feeler and brain after posture is fixed are placed under microscope and obtain different enlargement ratio inferior antennas
The image information of interior nerve fibre bundle adjusts the posture of feeler and fixation as needed, the image information of angle needed for obtaining.
The present invention using nickel chloride or cobalt chloride as Neural tracer, controlled by control tracer immerged time nickel from
The distance that son or cobalt ions enter the quantity of nerve and transmit backward guarantees that the activity of insect facilitates tracer during dip dyeing
The specificity of agent is disseminated, i.e., nickel ion or cobalt ions are travelled rearwardly only along aixs cylinder (nerve fibre bundle), and can be deposited in traveling
Distance in, as one of the committed step of tracer decides the result of colour developing.There is provided 50%-70%'s during dip dyeing
Humidity can reduce the evaporation of tracer aqueous solution, conducive to the osmotic pressure for maintaining tracer aqueous solution, reduce reactor solution to mind
Influence through cell；During microdissection, be filled with mixed gas to Ringer liquid and keep lower temperature also for
Guarantee the tissue of feeler and brain and the excellent activity of cell, it is non-to be diffused into other for tracer when preventing cell activity from reducing or is dead
In nerve fiber, subsequent color developing effect is influenced；Rubeanic acid can form darkviolet complex compound with nickel ion or cobalt ions, therefore
Darkviolet is dyed into the place that nickel ion or cobalt ions are accumulated, and then the nerve fibre bundle inside feeler is dyed into darkviolet,
The duration that colour developing is controlled during colour developing is most important, stops colour developing immediately after nerve fibre bundle is coloured in feeler, removes
It removes rubeane acid solution, and with alcohol washes, prevents excessive color development, as one of the committed step of tracer effect；Respectively with 5% He
10% formalin to after colour developing feeler and brain carry out tissue and fix；Again with 70%, 80%, 90% and 100% gradient
Alcohol is dehydrated；Gaultherolin is a kind of colorless and transparent liquid, and refractive index with higher is normally used for soft tissue
It is transparent；But since insect feeler cuticula is thicker, translucency is low, hinders the visualization of feeler internal structure.
It is found by many experiments, gaultherolin is capable of the cuticula ectoskeleton of transparent feeler light color to a certain extent.
Therefore, insect light color feeler ectoskeleton cuticula, feeler inner tissue and capsules of brain transparence are realized using gaultherolin；Opposition
The feeler or brain of body structure carry out various dimensions pose adjustment and fixation, and as depth obtains nerve fibre bundle image information in feeler
Key link, utilize the stone of solid irregular, translucency, different sizes than insect feeler smaller szie quartz sand
Sand ratio can combination manufacture different height fulcrum curved surface the characteristics of, realize to feeler posture it is any adjustment with fixation, together
When, it is low sticky to spend the unobstructed using the Auxiliary support, the optical path that play the fixation of feeler posture of high transparency gaultherolin and touch
The triple role of angle preservation thoroughly solves the problems, such as fixation, the permanent preservation of the small feeler of insect, has thoroughly removed inside feeler
The last obstacle of structures visualization link.
Claims (7)
1. a kind of method of nerve fibre bundle tracer in insect light color feeler, which is characterized in that specific step is as follows:
(1) living insects feeler carries out nickel chloride solution or cobalt chloride solution dip dyeing；
(2) the complete feeler of micro- separation living insects and brain；
(3) labeled to insect feeler and brain epineural fibre bundle using rubeane acid solution submergence insect feeler and brain, it absorbs red
Propylhomoserin solution, then the insect feeler and brain that are developed the color using alcohol washes；
(4) successively using the tissue in the insect feeler and brain after the fixed colour developing of formalin A and formalin B, then to insect
Feeler and brain are carried out dehydrating；
(5) insect feeler and brain are immersed in gaultherolin, handles 1~12h at room temperature；
(6) posture of insect feeler and brain is adjusted and fixed, then carries out photomicrograph and obtains under different angle, different enlargement ratios
The image information of nerve fibre bundle in feeler.
2. according to claim 1 in insect light color feeler nerve fibre bundle tracer method, it is characterised in that: step (1)
The mass concentration of middle nickel chloride solution or cobalt chloride solution is 1%~10%, if insect feeler length is n millimeters, then when disseminating
Between be n~n+5 hours.
3. according to claim 1 in insect light color feeler nerve fibre bundle tracer method, it is characterised in that: step (3)
Middle alcohol is absolute alcohol.
4. according to claim 1 in insect light color feeler nerve fibre bundle tracer method, it is characterised in that: step (4)
The mass concentration of formalin A is 5%, and the mass concentration of formalin B is 10%, and the formalin A set time is 10min,
The formalin B set time is 10min.
5. according to claim 1 in insect light color feeler nerve fibre bundle tracer method, it is characterised in that: step (4)
The specific method of dehydration be successively use mass concentration for 70%, 80%, 90%, 100% alcohol to insect feeler and
Insect brain is carried out dehydrating, and the dehydration of alcohol time of various concentration is 5min.
6. according to claim 1 in insect light color feeler nerve fibre bundle tracer method, it is characterised in that: step (6)
The posture of middle insect feeler and insect brain is fixed to use The Small Well device, and quartz sand is arranged in The Small Well device.
7. according to claim 6 in insect light color feeler nerve fibre bundle tracer method, it is characterised in that: quartz sand is
Two different sizes；The partial size of quartz sand is the 1/5-1/2 of sample minimum dimension, the mass ratio of two kinds of quartz sand be 1:(1~
3)。
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