CN110082380A - A method of it is connected with cell between In Situ Tem Study cell monolayer - Google Patents
A method of it is connected with cell between In Situ Tem Study cell monolayer Download PDFInfo
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- CN110082380A CN110082380A CN201910393442.XA CN201910393442A CN110082380A CN 110082380 A CN110082380 A CN 110082380A CN 201910393442 A CN201910393442 A CN 201910393442A CN 110082380 A CN110082380 A CN 110082380A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
- G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/07—Investigating materials by wave or particle radiation secondary emission
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/10—Different kinds of radiation or particles
- G01N2223/102—Different kinds of radiation or particles beta or electrons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/60—Specific applications or type of materials
- G01N2223/612—Specific applications or type of materials biological material
Abstract
The present invention relates to cell observation technical fields, the method connected more particularly, to cell between a kind of cell monolayer with In Situ Tem Study, the following steps are included: (1) cell prepares: will be put into culture plate by the epoxy sheet of sterilizing, cell monolayer is by centrifugation, resuspension, it is uniformly added into the substrate of epoxy sheet, is put into incubator and is cultivated；(2) sample is fixed, is dehydrated, is impregnated with；(3) marked region, repair block, pre- embedding；(4) pre- embedded block repairs block, re coating again；(5) be sliced, dye, Electronic Speculum observation: by above step, the present invention can reduce the original appearance of cell monolayer farthest, in situ.For conventional method, The present invention reduces the damages connected to cell；For external embedding in situ method, resin is simpler, more easy than with glass slide as the carrier that cell is grown, and the present invention embeds in advance and re coating method is more stable, effective.
Description
Technical field
The present invention relates to cell observation technical fields, more particularly, to a kind of between In Situ Tem Study cell monolayer
The method of cell connection.
Background technique
Cell connection is intercellular association structure, is formed by cytoplasma membrane regional area specialization, includes film in structure
Cytosolic fractions and the outer iuntercellular part of plasma membrane under specialization part, plasma membrane.In multicellular organisms, the bio-networks of cell connection are wide
General presence is connected each other by plasma membrane between flanking cell, and complicated biocommunicaion system is formd.Transmission electron microscope can be to original
Bit organization, culture cell, the intracellular and extracellular substances extracted in suspension cell carry out unique and valuable research.
The method for preparing transmission electron microscopy of traditional adhere-wall culture cell is: thin in pancreatin digestion or cell scraper scraping culture bottle
Born of the same parents are centrifuged into cell mass, are fixed, are dehydrated, are impregnated with, embed, are sliced, dye, and observe under last transmission electron microscope.Pancreatin disappears
Change makes iuntercellular junction protein degradation, and cell scraper scraping can generate mechanical injuries to cell, and both methods makes cell
Form and iuntercellular correlation change, and it is poor that cell connection effect is observed under transmission electron microscope.
The method for preparing transmission electron microscopy of traditional embedding in situ is: there is the loading back-off of cell in resin capsule culture,
Or directly resin capsule is buckled on cell culture container, capsule is cut into carry out sections observation after polymerization.Both methods
It is easy to appear the hole of Cells Depletion, when being impregnated with using embedding medium, cytomorphosis shrinkage is easy to cause, crack occurs, most
The observing effect of ultra microstructure is influenced eventually.
Home position observation cell monolayer method is under transmission electron microscope observing at present: being grown and is carried using glass cover-slip as cell
Body is fixed cell, is dehydrated, surface of glass slide resin embedding；It is with hydrofluoric acid that glass cover-slip is complete after standing a period of time
Full corrosion, resin embedding part are cut with diamond cut instrument, are stopped at cell level 1-2mm；Resin flake is placed in
On glass slide, circular target region is marked with permanent marker pen under optical microscopy, resin flake is placed in hot plate thereafter
1min carries out straight cuts into equilateral triangle to the region needed for studying in circular mark region；Take one No. 00
BEEMTMCapsule cuts away conical lower portion, forms open cylindrical body, and triangle resin flake is placed on bottom surface, drips tree
Rouge stands polymerization；By in the blocky resin after polymerization include original marked region part be cut into U-shaped, be disposed vertically in
BEEMTMOn capsule, resin is dripped, stands polymerization；Marked region is cut into quadrangularly, is cut into 600nm's with diamond cutter
Li Chasen dyestuff 1min is added in semithin section, cleans, thermal evaporation, room temperature preservation；It is cut into the slice of 100nm again, collects
In copper mesh, drying is placed in the culture dish of liner filter paper；Acetic acid uranium solution and lead citrate solution double staining rinse dry
It is observed under transmission electron microscope after net.This method is mainly the knot for being applied to home position observation schwann cells and schwann cells dorsal root ganglion
Structure, disadvantage is that: it uses glass slide as cellular growth support, can just leave tree after needing hydrofluoric acid attack glass
Rouge sample, hydrofluoric acid cell specimen to resin surface or can cause certain shadow to the cell connection of observation needed for the present invention
It rings；BEEMTMU-shaped vertically embeds operation and requires finely on capsule, and otherwise gravity is it is easier that resin embedding is incomplete；Sample needs
Will be by a glass attack, three times resin embedding, repeatedly cutting, consuming time is long, and required consumptive material is valuable, and step is many and diverse.
Therefore, a kind of morphology side that can in situ, intuitively, be effectively connected with transmission electron microscope observing cell is found
Method is most important.
The disclosure of background above technology contents is only used for auxiliary and understands inventive concept and technical solution of the invention, not
The prior art for necessarily belonging to present patent application shows above content in the applying date of present patent application in no tangible proof
In the case where having disclosed, above-mentioned background technique should not be taken to the novelty and creativeness of evaluation the application.
Summary of the invention
To solve the above-mentioned problems, the present invention, which provides one kind, can be reduced cellular damage, keep cell integrity to a greater extent
And the method for the cell connection that can be clearly observed between cell monolayer is urgently needed at present.
In order to realize the above problem, the technical solution adopted by the present invention are as follows:
A method of it is connected with cell between In Situ Tem Study cell monolayer, which is characterized in that including following step
It is rapid:
(1) cell prepares: will be put into culture plate by the Epon-812 epoxy sheet of sterilizing, cell monolayer is passed through
It crosses centrifugation, be resuspended, be uniformly added into the substrate of Epon-812 epoxy sheet, be put into incubator and cultivated；
(2) sample is fixed, is dehydrated, is impregnated with:
A, sample is fixed: glutaraldehyde fixer is added in the cell that step (1) is cultivated, and places 12-16h at 2-6 DEG C,
It is cleaned 3 times using 0.1mol/L phosphate buffer, each 8min；After reusing the fixed 1h of 1% osmic acid, 0.1mol/L phosphorus is used
Phthalate buffer cleans 3 times, each 8min；
B, ethyl alcohol is dehydrated step by step: 50% ethyl alcohol, 70% ethyl alcohol, 90% ethyl alcohol are dehydrated once 100% ethanol dehydration 3 respectively
It is secondary, the above each 8min of dehydration at different levels；
C, it is impregnated with: being impregnated with resin flake using Epon-812 epoxy resin, be put into 40 DEG C of baking oven 2h.
(3) marked region, repair block, pre- embedding:
A, cell is marked using marker pen to grow intensive region and marked region trimmed out to obtain cell intensive
Block,
B, pure Epon-812 epoxy resin prepolymer embedding: the two drop resin of drop in resin embedding plate in advance, the intensive block of cell
Cell close to embedding plate bottom end, eliminates extra bubble down, and makes the intensive block holding of entire cell horizontal, so that
Cell face is parallel with embedding plate bottom surface；
C, drip full resin embedding plate using pure Epon-812 epoxy resin embedding agent, be put into 40 DEG C, 48 DEG C, in 60 DEG C of baking ovens
It polymerize 15h, 12h, for 24 hours respectively.
(4) pre- embedded block repairs block, re coating again:
A, cell intensive block cell face in step (3) the pre- embedded block position parallel with bottom edge is marked, it is close to cell
Glomeration carries out polishing and is cut into cube resin fritter；
B, pure Epon-812 epoxy resin re coating: in advance in resin embedding plate drop two drop resins, will repair cut it is pre-
Embedded block is put into resin embedding plate, cell face outwardly and with embedding plate plane perpendicular；
C, drip full resin embedding plate using pure Epon-812 epoxy resin embedding agent, be put into 40 DEG C, 48 DEG C, in 60 DEG C of baking ovens
It polymerize 15h, 12h, for 24 hours respectively.
(5) it is sliced, dyes, Electronic Speculum observation:
A, the intensive block of the good cell of re coating is used into microtome, is cut into the semithin section with a thickness of 1um, dyeing is fixed
Position cellular layer, according to semithin section picture cues choose target area, centered on target area with diamond cutter cut with a thickness of
The slice of 70nm obtains complete cell monolayer section；
B, slice uses acetic acid uranium-lead citrate double staining, rinses, dries；
C, cell connection is observed under transmission electron microscope.
Further, Epon-812 epoxy sheet sterilization method described in the step (1) is double using ultraviolet lamp
Face irradiation.
Further, the cell monolayer is Human pancreatic ductal cell HPDE6C7, in the condition of culture of incubator
Are as follows: 37 DEG C, 5%CO2.。
Further, the cell density of the Human pancreatic ductal cell that plate is added be 1 × 10^6/
The hole ml/.
Further, the intensive a height of 3mm × 3mm of the block length and width × 0.5mm of cell in step (3) a.
The invention has the advantages that
1. between the In Situ Tem Study cell monolayer that the present invention constructs cell connect, use resin flake as
HPDE6C7 cellular growth support marks cell close quarters after fixation, being dehydrated, being impregnated under light microscopic, and trimming resin is thin
By embedding in advance, repairing block, re coating again after piece, the original appearance of cell monolayer is reduced farthest, in situ.It compares
For conventional method, The present invention reduces the damages connected to cell；For embedding in situ method, resin is as thin
The carrier of intracellular growth is simpler, more easy than with glass slide, and the present invention embeds in advance and re coating method is more stable, has
Effect can more clearly observe the cell connection between cell monolayer.
2. the cell of single layer cells in situ can be made to face outwardly vertical stand-up, cut in this way by embedding and re coating in advance
Available one complete cell plane when cutting.
Detailed description of the invention
Fig. 1 is HPDE6C7 cell of the present invention in Epon-812 epoxy sheet cultivation situation；
Fig. 2 is HPDE6C7 cell of the present invention marked situation on six orifice plates；
Fig. 3 is that the method for the present invention observes Cell tracking schematic diagram under transmission electron microscope；
Fig. 4 is that the method for the present invention observes Cell tracking schematic diagram under transmission electron microscope；
Fig. 5 is that the method for the present invention observes Cell tracking schematic diagram under transmission electron microscope；
Fig. 6 is that the method for the present invention observes Cell tracking schematic diagram under transmission electron microscope；
Fig. 7 is that the method for the present invention observes Cell tracking schematic diagram under transmission electron microscope；
Fig. 8 is that the method for the present invention observes Cell tracking schematic diagram under transmission electron microscope.
Specific embodiment
Invention is further described in detail With reference to embodiment.It is emphasized that following the description is only
It is merely exemplary, the range and its application being not intended to be limiting of the invention.
Embodiment 1
Material prepares: cell and its growing carrier: Human pancreatic ductal cell strain (HPDE6C7), and 15mm × 20mm ×
The Epon-812 epoxy sheet of 0.5mm.
Cell prepares: (1) HPDE6C7 cell growth state is good, covers about 90%T25 culture bottle；(2) will pass through ultraviolet
The Epon-812 epoxy sheet of lamp double-sided illumination is put into culture plate；(3) 0.25% trypsin digestion cells, centrifugation, resuspension,
Cell count is homogeneously added into six orifice plates of resin flake with 1 × 10^6/hole ml/ cell density, be put into 37 DEG C,
5%CO2Incubator culture.
Sample is fixed, is dehydrated, is impregnated with: (1) covering 90% or more resin flake to cell, 3% glutaraldehyde is added and fixes
Liquid, 4 DEG C of placement 12-16h；Show that HPDE6C7 cell is cultivated in order in Epon-812 epoxy sheet as shown in Figure 1；
(2) 0.1mol/L phosphate buffer (PBS) cleans 3 times, each 8min；After the fixed 1h of (3) 1% osmic acids, 0.1mol/L phosphoric acid
Salt buffer (PBS) cleans 3 times, each 8min；(4) ethyl alcohol is dehydrated step by step: 50% ethyl alcohol, 70% ethyl alcohol, 90% ethyl alcohol difference
Dehydration is primary, 100% ethanol dehydration 3 times, the above each 8min of dehydration at different levels；(5) it is impregnated with using pure Epon-812 epoxy resin
Resin flake is put into 40 DEG C of baking oven 2h.
Marked region repairs block, pre- embedding: (1) observing resin flake with 4 × eyepiece under optical microscopy, existed with marker pen
Cell is marked on six orifice plates and grows intensive region, and HPDE6C7 cell as shown in Figure 2 marked situation on six orifice plates shows
HPDE6C7 cell is cultivated in order in Epon-812 epoxy sheet；(2) total with eye scissors, ophthalmic tweezers and single-edge blade
Marked region is trimmed to the intensive block of 3mm × 3mm × 0.5mm cell, pays attention to distinguishing cell face and acellular face by biconditional operation；
(3) pure Epon-812 epoxy resin prepolymer embedding: two drop resin of drop, the corresponding paper labels face of sample in resin embedding plate in advance
Upward, the cell of the intensive block of cell is face-down, and extra bubble is chosen away with toothpick, notices that the intensive block of entire cell will be protected as far as possible
Water holding is flat, so that cell face is parallel with embedding plate bottom surface, in order to subsequent operation；(4) pure Epon-812 epoxy resin embedding agent
Drip full resin embedding plate, be put into 40 DEG C, 48 DEG C, polymerize 15h, 12h, for 24 hours in 60 DEG C of baking ovens respectively.
Pre- embedded block repairs block, re coating again: (1) marker pen marks the intensive block of cell in pre- embedded block under table magnifier
The cell face position parallel with bottom edge is polished with STRONG90 electric grinding machine, is aided with small-sized holding hacksaw and is repaired that be cut into diagram vertical
Cube resin fritter；(2) pure Epon-812 epoxy resin re coating: two drop resin of drop, sample pair in resin embedding plate in advance
The paper labels answered are face-up, will repair the pre- embedded block cut and are put into resin embedding plate, notice that cell faces outwardly and and embedding plate
Full resin embedding plate is dripped in plane perpendicular, pure Epon-812 epoxy resin embedding agent, be put into 40 DEG C, 48 DEG C, in 60 DEG C of baking ovens respectively
It polymerize 15h, 12h, for 24 hours.
Slice, dyeing, Electronic Speculum observation: (1) coming card EM UC7 ultramicrotome and be sliced, and cutting thickness is about the half thinly-sliced of 1um
Piece, dyeing positioning cellular layer, is chosen target area according to semithin section picture cues, is cut centered on target area with diamond cutter
With a thickness of the ultra-thin section of 70nm, noticing when slice that technical staff will adjust the angle keeps knife section vertical with cell face, thus must
To complete cell monolayer section；(2) obtained serial section is collected into copper mesh, is placed on the culture dish for being covered with filter paper
In, corresponding sample label can be marked on filter paper with pencil；(3) acetic acid uranium-lead citrate double staining, rinses, dries；
(4) cell connection is observed under Hitachi H-7650 transmission electron microscope, as shown in figures 3-8 visible clearly cell connection, packet under Electronic Speculum
Include the various cell connection types such as cell connection, gap connection, close connection, desmosome, intermediate connection.
Embodiment 2
(1) cell prepares: HPDE6C7 cell growth state is good, covers about 90%T25 culture bottle；(2) will pass through ultraviolet
The Epon-812 epoxy sheet of lamp double-sided illumination is put into culture plate；(3) 0.25% trypsin digestion cells, centrifugation, resuspension,
Cell count is homogeneously added into six orifice plates of resin flake with 1 × 10^6/hole ml/ cell density, be put into 37 DEG C,
5%CO2Incubator culture；
(2) sample is fixed, is dehydrated, is impregnated with:
A, sample is fixed: glutaraldehyde fixer is added in the cell that step (1) is cultivated, and 12h is placed at 4 DEG C, uses
0.1mol/L phosphate buffer cleans 3 times, each 8min；After reusing the fixed 1h of 1% osmic acid, 0.1mol/L phosphate is used
Buffer solution for cleaning 3 times, each 8min；
B, ethyl alcohol is dehydrated step by step: 50% ethyl alcohol, 70% ethyl alcohol, 90% ethyl alcohol are dehydrated once 100% ethanol dehydration 3 respectively
It is secondary, the above each 8min of dehydration at different levels；
C, it is impregnated with: being impregnated with resin flake using Epon-812 epoxy resin, be put into 40 DEG C of baking oven 2h.
(3) marked region, repair block, pre- embedding:
A, cell is marked using marker pen grow intensive region and trim out by marked region obtain 3mm × 3mm
The intensive block of × 0.5mm cell；
B, pure Epon-812 epoxy resin prepolymer embedding: the two drop resin of drop in resin embedding plate in advance, the intensive block of cell
Cell close to embedding plate bottom end, eliminates extra bubble down, and makes the intensive block holding of entire cell horizontal, so that
Cell face is parallel with embedding plate bottom surface；
C, drip full resin embedding plate using pure Epon-812 epoxy resin embedding agent, be put into 40 DEG C, 48 DEG C, in 60 DEG C of baking ovens
It polymerize 15h, 12h, for 24 hours respectively.
(4) pre- embedded block repairs block, re coating again:
A, cell intensive block cell face in step (3) the pre- embedded block position parallel with bottom edge is marked, it is close to cell
Glomeration carries out polishing and is cut into cube resin fritter；
B, pure Epon-812 epoxy resin re coating: in advance in resin embedding plate drop two drop resins, will repair cut it is pre-
Embedded block is put into resin embedding plate, cell face outwardly and with embedding plate plane perpendicular；
C, drip full resin embedding plate using pure Epon-812 epoxy resin embedding agent, be put into 40 DEG C, 48 DEG C, in 60 DEG C of baking ovens
It polymerize 15h, 12h, for 24 hours respectively.
(5) it is sliced, dyes, Electronic Speculum observation:
A, the intensive block use of the good cell of re coating is come into card EM UC7 ultramicrotome slice, be cut into a thickness of 1um's
Semithin section, dyeing positioning cellular layer, chooses target area according to semithin section picture cues, with brill centered on target area
The slice that stone knife is cut with a thickness of 70nm obtains complete cell monolayer section；
B, slice uses acetic acid uranium-lead citrate double staining, rinses, dries；
C, cell connection is observed under Hitachi H-7650 transmission electron microscope.
The above content is combine it is specific/further detailed description of the invention for preferred embodiment, cannot
Assert that specific implementation of the invention is only limited to these instructions.General technical staff of the technical field of the invention is come
It says, without departing from the inventive concept of the premise, some replacements or modifications can also be made to the embodiment that these have been described,
And these substitutions or variant are regarded as belonging to the scope of protection of the present invention.
Claims (5)
1. a kind of method that cell connects between cell monolayer with In Situ Tem Study, which comprises the following steps:
(1) cell prepare: will by sterilizing Epon-812 epoxy sheet be put into culture plate, by cell monolayer pass through from
The heart, resuspension are uniformly added into the substrate of Epon-812 epoxy sheet, are put into incubator and are cultivated；
(2) sample is fixed, is dehydrated, is impregnated with:
A, sample is fixed: glutaraldehyde fixer is added in the cell that step (1) is cultivated, and 12h is placed at 4 DEG C, uses
0.1mol/L phosphate buffer cleans 3 times, each 8min；After reusing the fixed 1h of 1% osmic acid, 0.1mol/L phosphate is used
Buffer solution for cleaning 3 times, each 8min；
B, ethyl alcohol is dehydrated step by step: 50% ethyl alcohol, 70% ethyl alcohol, 90% ethyl alcohol are dehydrated once 100% ethanol dehydration 3 times respectively, with
The upper each 8min of dehydration at different levels；
C, it is impregnated with: being impregnated with resin flake using pure Epon-812 epoxy resin, be put into 40 DEG C of baking oven 2h；
(3) marked region, repair block, pre- embedding:
A, cell is marked using marker pen grow intensive region and trim out by marked region obtain the intensive block of cell；
B, pure Epon-812 epoxy resin prepolymer embedding: two drop resin of drop, the cell of the intensive block of cell in resin embedding plate in advance
Down close to embedding plate bottom end, extra bubble is eliminated, and makes the intensive block holding of entire cell horizontal, so that cell
Face is parallel with embedding plate bottom surface；
C, drip full resin embedding plate using pure Epon-812 epoxy resin embedding agent, be put into 40 DEG C, 48 DEG C, in 60 DEG C of baking ovens respectively
It polymerize 15h, 12h, for 24 hours；
(4) pre- embedded block repairs block, re coating again:
A, cell intensive block cell face in step (3) the pre- embedded block position parallel with bottom edge is marked, to the intensive block of cell
It carries out polishing and is cut into cube resin fritter；
B, pure Epon-812 epoxy resin re coating: the two drop resin of drop in resin embedding plate in advance will repair the pre- embedding cut
Block is put into resin embedding plate, cell face outwardly and with embedding plate plane perpendicular；
C, drip full resin embedding plate using pure Epon-812 epoxy resin embedding agent, be put into 40 DEG C, 48 DEG C, in 60 DEG C of baking ovens respectively
It polymerize 15h, 12h, for 24 hours.
(5) it is sliced, dyes, Electronic Speculum observation:
A, the intensive block of the good cell of re coating is used into microtome, is cut into the semithin section with a thickness of 1um, dyeing positioning is thin
Born of the same parents' layer is chosen target area according to semithin section picture cues, is cut centered on target area with diamond cutter with a thickness of 70nm's
Slice obtains complete cell monolayer section；
B, slice uses acetic acid uranium-lead citrate double staining, rinses, dries；
C, cell connection is observed under transmission electron microscope.
2. the method connected according to claim 1 with cell between In Situ Tem Study cell monolayer, it is characterised in that:
The Epon-812 epoxy sheet mode of sterilizing described in the step (1) is to use ultraviolet lamp double-sided illumination.
3. the method connected according to claim 1 with cell between In Situ Tem Study cell monolayer, it is characterised in that:
The cell monolayer is Human pancreatic ductal cell HPDE6C7, in the condition of culture of incubator are as follows: 37 DEG C, 5%CO2.。
4. the method connected according to claim 3 with cell between In Situ Tem Study cell monolayer, it is characterised in that:
The cell density of the Human pancreatic ductal cell that plate is added is the 1 × 10^6/hole ml/.
5. the method connected according to claim 4 with cell between In Situ Tem Study cell monolayer, it is characterised in that:
The intensive a height of 3mm × 3mm of the block length and width × 0.5mm of cell in step (3) a.
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| CN113109114A (en) * | 2021-04-14 | 2021-07-13 | 中山大学 | Pretreatment method for analyzing rare earth elements in apatite and detection method thereof |
| CN113640323A (en) * | 2021-09-07 | 2021-11-12 | 南通大学 | Preparation method of transmission electron microscope sample with small number of cells |
| CN113654863A (en) * | 2021-08-30 | 2021-11-16 | 南通大学 | Sample processing method of in-situ in-vitro cultured cells for transmission electron microscope observation |
| CN113933138A (en) * | 2020-07-14 | 2022-01-14 | 西北农林科技大学 | Transmission electron microscope embedding method |
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| CN113109114A (en) * | 2021-04-14 | 2021-07-13 | 中山大学 | Pretreatment method for analyzing rare earth elements in apatite and detection method thereof |
| CN113654863A (en) * | 2021-08-30 | 2021-11-16 | 南通大学 | Sample processing method of in-situ in-vitro cultured cells for transmission electron microscope observation |
| WO2023029909A1 (en) * | 2021-08-30 | 2023-03-09 | 南通大学 | Sample treatment method for in-situ in vitro culture cells for observation by transmission electron microscopy |
| CN113640323A (en) * | 2021-09-07 | 2021-11-12 | 南通大学 | Preparation method of transmission electron microscope sample with small number of cells |
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