CN110082380B - Method for in-situ observation of intercellular cell junction of single layer by transmission electron microscope - Google Patents
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Abstract
The invention relates to the technical field of cell observation, in particular to a method for in-situ observation of intercellular cell junction of a single layer by using a transmission electron microscope, which comprises the following steps: (1) cell preparation: placing the sterilized epoxy resin slices into a culture plate, centrifuging and resuspending monolayer cells, uniformly adding the monolayer cells into a substrate with the epoxy resin slices, and placing the substrate into an incubator for culture; (2) fixing, dehydrating and soaking the specimen; (3) marking a region, repairing a block and pre-embedding; (4) repairing and re-embedding the pre-embedded blocks; (5) slicing, dyeing and observing by an electron microscope: through the steps, the invention can reduce the original appearance of the monolayer cells to the maximum extent and in situ. Compared with the traditional method, the invention reduces the damage to cell connection; compared with the foreign in-situ embedding method, the resin is simpler and easier to be used as a carrier for cell growth than a glass slide glass, and the pre-embedding and re-embedding method is more stable and effective.
Description
Technical Field
The invention relates to the technical field of cell observation, in particular to a method for in-situ observation of intercellular cell junction of a single layer by using a transmission electron microscope.
Background
Cell junctions are intercellular communication structures formed by the specification of local regions of the plasma membrane of cells, structurally including membrane-specific portions, plasma membrane subcoplasmic portions, and extraplasma membrane intercellular portions. In multicellular organisms, biological networks of cell junctions are widely present, and adjacent cells are interconnected through plasma membranes, thereby forming a complex biological communication system. Transmission electron microscopy enables unique and valuable research into intracellular and extracellular material extracted from in situ tissues, cultured cells, and suspension cells.
The transmission electron microscope sample preparation method of the traditional adherent culture cell comprises the following steps: pancreatin digestion or cell scraping to obtain cell mass, fixing, dewatering, soaking, embedding, slicing, dyeing and observing under transmission electron microscope. The pancreatin digestion degrades protein at the joint between cells, and the scraping of the cells can cause mechanical damage to the cells, so that the cell morphology and the intercellular interrelation are changed by the two methods, and the cell connection effect observed under a transmission electron microscope is poor.
The traditional in-situ embedded transmission electron microscope sample preparation method comprises the following steps: and (3) inverting the object with the cultured cells on a resin capsule, or directly inverting the resin capsule on a cell culture container, and cutting the capsule after polymerization for slicing and observation. Both methods are easy to generate holes for cell deletion, and when the embedding agent is soaked and used, cells are easy to deform and shrink and generate cracks, so that the observation effect of the ultrastructure is influenced finally.
The prior method for observing the monolayer cells in situ under the observation of a transmission electron microscope comprises the following steps: taking a glass cover glass as a cell growth carrier, fixing and dehydrating cells, and embedding the surface of the glass slide with resin; standing for a period of time, completely corroding the glass cover glass with hydrofluoric acid, cutting the resin-embedded part with a diamond cutting instrument, and stopping at a position 1-2mm away from the cell layer; placing the resin slice on a glass slide, marking a circular target area by using a permanent marking pen under an optical microscope, then placing the resin slice on a hot plate for 1min, and linearly cutting an area needing to be used for research into an equilateral triangle in the circular marking area; taking a BEEM No. 00TMEncapsulating, cutting off the conical bottom to form an open cylinder, placing a triangular resin sheet on the bottom surface, dripping a drop of resin, and standing for polymerization; cutting the polymerized block resin including the original mark region into U shape, and vertically placing in BEEMTMDripping a drop of resin on the capsule, standing and polymerizing; cutting the marked area into quadrangles, cutting into 600nm semi-thin slices by using a diamond knife, adding Richardson dye for 1min, cleaning, thermally evaporating, and storing at room temperature; cutting into thin slices of 100nm, collecting on copper grid, drying, and placing in a culture dish lined with filter paper; vinegarAnd carrying out double dyeing on the uranium acid solution and the lead citrate solution, and observing under a transmission electron microscope after the uranium acid solution and the lead citrate solution are washed clean. The method is mainly applied to in-situ observation of Schwann cells and the structures of Schwann cell dorsal root ganglia, and has the following defects: the glass slide is used as a cell growth carrier, a resin specimen can be reserved only after the glass is corroded by hydrofluoric acid, and the hydrofluoric acid can cause certain influence on the cell specimen on the surface of the resin or cell connection required to be observed by the invention; BEEMTMThe U-shaped vertical embedding operation on the capsule requires fine operation, otherwise the resin is easy to be incompletely embedded under the action of gravity; the specimen needs to be subjected to one-time glass erosion, three-time resin embedding and multiple cutting, the consumed time is long, the required consumables are expensive, and the steps are complicated.
Therefore, it is important to find a morphological method capable of in situ, intuitively and effectively observing cell junctions by transmission electron microscopy.
The above background disclosure is only for the purpose of assisting understanding of the inventive concept and technical solutions of the present invention, and does not necessarily belong to the prior art of the present patent application, and should not be used for evaluating the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.
Disclosure of Invention
In order to solve the above problems, it is desirable to provide a method for reducing cell damage, maintaining cell integrity to a greater extent, and clearly observing cell junctions between cells in a monolayer.
In order to realize the problems, the invention adopts the technical scheme that:
a method for in-situ observation of intercellular cell junctions of a single layer by using a transmission electron microscope is characterized by comprising the following steps:
(1) cell preparation: placing the sterilized Epon-812 epoxy resin slices into a culture plate, centrifuging and resuspending single-layer cells, uniformly adding the single-layer cells into a substrate with the Epon-812 epoxy resin slices, and placing the substrate into an incubator for culture;
(2) fixing, dehydrating and soaking a specimen:
a. fixing a specimen: adding the cells cultured in the step (1) into glutaraldehyde stationary liquid, standing at 2-6 ℃ for 12-16h, and washing for 8min each time for 3 times by using 0.1mol/L phosphate buffer solution; after further fixation with 1% osmic acid for 1h, washing with 0.1mol/L phosphate buffer solution for 3 times, each time for 8 min;
b. and (3) dehydrating ethanol step by step: dehydrating 50% ethanol, 70% ethanol and 90% ethanol respectively for one time, dehydrating 100% ethanol for 3 times, and dehydrating above each stage for 8 min;
c. soaking: the resin sheet was impregnated with Epon-812 epoxy resin and placed in a 40 ℃ oven for 2 h.
(3) Marking areas, repairing blocks and pre-embedding:
a. marking the areas with dense cell growth by using a marking pen and trimming the marked areas to obtain dense cell blocks,
b. pure Epon-812 epoxy pre-embedded: dripping two drops of resin in the resin embedding plate in advance, wherein the cell surface of the cell dense block is downward and is close to one end of the bottom of the embedding plate, eliminating redundant bubbles, and keeping the whole cell dense block horizontal so that the cell surface is parallel to the bottom of the embedding plate;
c. the resin embedding plate is fully dripped with pure Epon-812 epoxy resin embedding agent, and is put into an oven with the temperature of 40 ℃, 48 ℃ and 60 ℃ for polymerization for 15h, 12h and 24h respectively.
(4) Repairing the pre-embedded block and re-embedding:
a. marking the part, parallel to the bottom edge, of the cell surface of the cell dense block in the pre-embedded block in the step (3), and polishing and cutting the cell dense block into cubic resin small blocks;
b. pure Epon-812 epoxy resin re-embedding: dripping two drops of resin in the resin embedding plate in advance, putting the trimmed pre-embedding block into the resin embedding plate, and enabling the cell surface to be outward and vertical to the bottom surface of the embedding plate;
c. the resin embedding plate is fully dripped with pure Epon-812 epoxy resin embedding agent, and is put into an oven with the temperature of 40 ℃, 48 ℃ and 60 ℃ for polymerization for 15h, 12h and 24h respectively.
(5) Slicing, dyeing and observing by an electron microscope:
a. slicing the heavily-embedded cell dense block by using a slicer, cutting into a semi-thin slice with the thickness of 1um, staining and positioning a cell layer, selecting a target area according to the image prompt of the semi-thin slice, and cutting the slice with the thickness of 70nm by using a diamond knife by taking the target area as the center to obtain a complete single-layer cell section;
b. the slice is dyed by uranium acetate-lead citrate, washed and dried;
c. cell junctions were observed under transmission electron microscopy.
Further, in the step (1), the Epon-812 epoxy resin sheet is sterilized by double-sided irradiation with ultraviolet lamps.
Further, the monolayer cells are human pancreatic ductal epithelial cells HPDE6C7 cultured in a culture box at 37 deg.C and 5% CO2.。
Further, the cell density of the human pancreatic ductal epithelial cells added to the culture substrate is 1 x 10^ 6/ml/well.
Further, the length and width of the cell dense block in the step (3) a are 3mm × 3mm × 0.5 mm.
The beneficial effect of the invention is that,
1. the transmission electron microscope constructed by the invention observes the cell connection among the monolayer cells in situ, uses a resin sheet as an HPDE6C7 cell growth carrier, marks the cell dense area under the light mirror after fixing, dehydrating and soaking, and reduces the original appearance of the monolayer cells to the maximum extent in situ through the steps of pre-embedding, re-blocking, re-embedding and the like after trimming the resin sheet. Compared with the traditional method, the invention reduces the damage to cell connection; compared with the in-situ embedding method, the resin is simpler and easier to be used as a carrier for cell growth than a glass slide glass, and the pre-embedding and re-embedding method is more stable and effective, and can more clearly observe the cell connection between single-layer cells.
2. By pre-embedding and re-embedding, the cell surface of the monolayer in situ cells can be enabled to be vertically upright outwards, so that a complete cell plane can be obtained during cutting.
Drawings
FIG. 1 shows the conditions of the present invention in which HPDE6C7 cells were cultured on Epon-812 epoxy resin sheets;
FIG. 2 shows the labeling of HPDE6C7 cells of the present invention on a six-well plate;
FIG. 3 is a schematic diagram of intercellular junctions observed under a transmission electron microscope according to the method of the present invention;
FIG. 4 is a schematic diagram of intercellular junctions observed under a transmission electron microscope according to the method of the present invention;
FIG. 5 is a schematic diagram of intercellular junctions observed under a transmission electron microscope according to the method of the present invention;
FIG. 6 is a schematic diagram of intercellular junctions observed under a transmission electron microscope according to the method of the present invention;
FIG. 7 is a schematic diagram of intercellular junctions observed under a transmission electron microscope according to the method of the present invention;
FIG. 8 is a schematic diagram of intercellular junctions observed under a transmission electron microscope according to the method of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
Example 1
Preparing materials: cells and their growth vectors: human pancreatic ductal epithelial cell line (HPDE6C7), Epon-812 epoxy sheet 15mm by 20mm by 0.5 mm.
Cell preparation: (1) the growth state of HPDE6C7 cells is good, and the HPDE6C7 cells cover about 90% of T25 culture bottles; (2) placing Epon-812 epoxy resin slices subjected to double-sided irradiation by an ultraviolet lamp into a culture plate; (3) 0.25% pancreatin digestion of cells, centrifugation, resuspension, cell counting, adding into six-well plate with resin sheet at cell density of 1 × 10^ 6/ml/well, placing at 37 deg.C with 5% CO2 ofCulturing in an incubator.
Fixing, dehydrating and soaking a specimen: (1) adding 3% glutaraldehyde fixing solution when the resin sheet is covered by more than 90% of cells, and standing at 4 ℃ for 12-16 h; as shown in FIG. 1, HPDE6C7 cells were well cultured on Epon-812 epoxy resin sheets; (2) washing with 0.1mol/L Phosphate Buffer Solution (PBS) for 3 times, each time for 8 min; (3) after 1% osmic acid fixation for 1h, 0.1mol/L Phosphate Buffered Saline (PBS) was washed 3 times for 8min each; (4) and (3) dehydrating ethanol step by step: dehydrating 50% ethanol, 70% ethanol and 90% ethanol respectively for one time, dehydrating 100% ethanol for 3 times, and dehydrating above each stage for 8 min; (5) the resin sheets were impregnated with pure Epon-812 epoxy resin and placed in a 40 ℃ oven for 2 h.
Marking areas, repairing blocks and pre-embedding: (1) observing the resin slice by using a 4 Xeyepiece under an optical microscope, and marking a region with dense cell growth on a six-well plate by using a marking pen, wherein the marking condition of the HPDE6C7 cells on the six-well plate shown in figure 2 shows that the culturing condition of the HPDE6C7 cells on the Epon-812 epoxy resin slice is good; (2) using an ophthalmic scissors, an ophthalmic forceps and a single-sided blade to operate together, trimming the marked region into a cell dense block with the size of 3mm multiplied by 0.5mm, and paying attention to the cell surface and the non-cell surface; (3) pure Epon-812 epoxy pre-embedded: dripping two drops of resin in a resin embedding plate in advance, enabling a paper label corresponding to a sample to face upwards and a cell surface of a cell dense block to face downwards, picking away redundant bubbles by using a toothpick, and paying attention to the fact that the whole cell dense block is kept horizontal as much as possible to enable the cell surface to be parallel to the bottom surface of the embedding plate so as to facilitate subsequent operation; (4) the pure Epon-812 epoxy resin embedding agent is dripped into the resin embedding plate, and the resin embedding plate is put into an oven with the temperature of 40 ℃, 48 ℃ and 60 ℃ for polymerization for 15h, 12h and 24h respectively.
Repairing the pre-embedded block and re-embedding: (1) marking the parallel part of the cell surface and the bottom edge of the cell dense block in the pre-embedded block by a marking pen under a table type magnifier, polishing by using a STRONG90 electric polisher, and trimming into small display cubic resin blocks by using a small hand-held steel saw; (2) pure Epon-812 epoxy resin re-embedding: dropping two drops of resin in a resin embedding plate in advance, enabling the paper label corresponding to a sample to face upwards, placing the trimmed pre-embedding block into the resin embedding plate, paying attention to that the cell face faces outwards and is vertical to the bottom surface of the embedding plate, dropping pure Epon-812 epoxy resin embedding agent into the resin embedding plate, and placing the resin embedding plate into an oven at 40 ℃, 48 ℃ and 60 ℃ for polymerization for 15 hours, 12 hours and 24 hours respectively.
Slicing, dyeing and observing by an electron microscope: (1) the Leica EM UC7 ultrathin slicer slices, cuts a semi-thin slice with the thickness of about 1um, dyes and positions a cell layer, selects a target area according to the prompting of a semi-thin slice image, cuts the ultrathin slice with the thickness of 70nm by using a diamond knife with the target area as the center, and pays attention to that technicians need to adjust the angle to ensure that the knife section is vertical to a cell plane when slicing so as to obtain a complete single-layer cell section; (2) collecting the obtained continuous slices on a copper grid, placing the copper grid on a culture dish paved with filter paper, and marking corresponding sample labels on the filter paper by using a pencil; (3) carrying out double dyeing on uranium acetate-lead citrate, washing and airing; (4) cell junctions were observed under a transmission electron microscope, and clear cell junctions including cell junctions, gap junctions, tight junctions, desmosomes, and intermediate junctions were observed under the transmission electron microscope as shown in FIGS. 3-8.
Example 2
(1) Cell preparation: the growth state of HPDE6C7 cells is good, and the HPDE6C7 cells cover about 90% of T25 culture bottles; (2) placing Epon-812 epoxy resin slices subjected to double-sided irradiation by an ultraviolet lamp into a culture plate; (3) 0.25% pancreatin digestion of cells, centrifugation, resuspension, cell counting, adding into six-well plate with resin sheet at cell density of 1 × 10^ 6/ml/well, placing at 37 deg.C with 5% CO2 ofCulturing in an incubator;
(2) fixing, dehydrating and soaking a specimen:
a. fixing a specimen: adding the cells cultured in the step (1) into glutaraldehyde stationary liquid, standing at 4 ℃ for 12h, and washing for 3 times with 0.1mol/L phosphate buffer solution for 8min each time; after further fixation with 1% osmic acid for 1h, washing with 0.1mol/L phosphate buffer solution for 3 times, each time for 8 min;
b. and (3) dehydrating ethanol step by step: dehydrating 50% ethanol, 70% ethanol and 90% ethanol respectively for one time, dehydrating 100% ethanol for 3 times, and dehydrating above each stage for 8 min;
c. soaking: the resin sheet was impregnated with Epon-812 epoxy resin and placed in a 40 ℃ oven for 2 h.
(3) Marking areas, repairing blocks and pre-embedding:
a. marking the areas with dense cell growth by using a marking pen and trimming the marked areas to obtain cell dense blocks of 3mm multiplied by 0.5 mm;
b. pure Epon-812 epoxy pre-embedded: dripping two drops of resin in the resin embedding plate in advance, wherein the cell surface of the cell dense block is downward and is close to one end of the bottom of the embedding plate, eliminating redundant bubbles, and keeping the whole cell dense block horizontal so that the cell surface is parallel to the bottom of the embedding plate;
c. the resin embedding plate is fully dripped with pure Epon-812 epoxy resin embedding agent, and is put into an oven with the temperature of 40 ℃, 48 ℃ and 60 ℃ for polymerization for 15h, 12h and 24h respectively.
(4) Repairing the pre-embedded block and re-embedding:
a. marking the part, parallel to the bottom edge, of the cell surface of the cell dense block in the pre-embedded block in the step (3), and polishing and cutting the cell dense block into cubic resin small blocks;
b. pure Epon-812 epoxy resin re-embedding: dripping two drops of resin in the resin embedding plate in advance, putting the trimmed pre-embedding block into the resin embedding plate, and enabling the cell surface to be outward and vertical to the bottom surface of the embedding plate;
c. the resin embedding plate is fully dripped with pure Epon-812 epoxy resin embedding agent, and is put into an oven with the temperature of 40 ℃, 48 ℃ and 60 ℃ for polymerization for 15h, 12h and 24h respectively.
(5) Slicing, dyeing and observing by an electron microscope:
a. slicing the heavily-embedded cell dense block by using a come EM UC7 ultrathin slicer, cutting into a semi-thin slice with the thickness of 1um, dyeing and positioning a cell layer, selecting a target area according to the image prompt of the semi-thin slice, and cutting a slice with the thickness of 70nm by using a diamond knife with the target area as the center to obtain a complete single-layer cell section;
b. the slice is dyed by uranium acetate-lead citrate, washed and dried;
c. cell junctions were observed under Hitachi H-7650 transmission electron microscopy.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and such substitutions and modifications are to be considered as within the scope of the invention.
Claims (5)
1. A method for in-situ observation of intercellular cell junctions of a single layer by using a transmission electron microscope is characterized by comprising the following steps:
(1) cell preparation: placing the sterilized Epon-812 epoxy resin slices into a culture plate, centrifuging and resuspending single-layer cells, uniformly adding the single-layer cells into a substrate with the Epon-812 epoxy resin slices, and placing the substrate into a culture box for culture, wherein the single-layer cells are human pancreatic ductal epithelial cells HPDE6C 7;
(2) fixing, dehydrating and soaking a specimen:
a. fixing a specimen: adding the cells cultured in the step (1) into glutaraldehyde stationary liquid, standing at 4 ℃ for 12h, and washing for 3 times with 0.1mol/L phosphate buffer solution for 8min each time; after further fixation with 1% osmic acid for 1h, washing with 0.1mol/L phosphate buffer solution for 3 times, each time for 8 min;
b. and (3) dehydrating ethanol step by step: dehydrating 50% ethanol, 70% ethanol and 90% ethanol respectively for one time, dehydrating 100% ethanol for 3 times, and dehydrating above each stage for 8 min;
c. soaking: soaking the resin sheet with pure Epon-812 epoxy resin, and placing the resin sheet in an oven at 40 ℃ for 2 h;
(3) marking areas, repairing blocks and pre-embedding:
a. marking a region with dense cell growth by using a marking pen and trimming the marked region to obtain a cell dense block;
b. pure Epon-812 epoxy pre-embedded: dripping two drops of resin in the resin embedding plate in advance, wherein the cell surface of the cell dense block is downward and is close to one end of the bottom of the embedding plate, eliminating redundant bubbles, and keeping the whole cell dense block horizontal so that the cell surface is parallel to the bottom of the embedding plate;
c. dripping pure Epon-812 epoxy resin embedding agent into the resin embedding plate, and respectively polymerizing for 15h, 12h and 24h in an oven at 40 ℃, 48 ℃ and 60 ℃;
(4) repairing the pre-embedded block and re-embedding:
a. marking the part, parallel to the bottom edge, of the cell surface of the cell dense block in the pre-embedded block in the step (3), and polishing and cutting the cell dense block into cubic resin small blocks;
b. pure Epon-812 epoxy resin re-embedding: dripping two drops of resin in the resin embedding plate in advance, putting the trimmed pre-embedding block into the resin embedding plate, and enabling the cell surface to be outward and vertical to the bottom surface of the embedding plate;
c. dripping pure Epon-812 epoxy resin embedding agent into the resin embedding plate, and respectively polymerizing for 15h, 12h and 24h in an oven at 40 ℃, 48 ℃ and 60 ℃;
(5) slicing, dyeing and observing by an electron microscope:
a. slicing the heavily-embedded cell dense block by using a slicer, cutting the cell dense block into a semi-thin slice with the thickness of 1um, dyeing and positioning a cell layer, selecting a target area according to the image prompt of the semi-thin slice, and cutting a thin slice with the thickness of 70nm by using a diamond knife by taking the target area as the center to obtain a complete single-layer cell section;
b. the slice is dyed by uranium acetate-lead citrate, washed and dried;
c. cell junctions were observed under transmission electron microscopy.
2. The method for in situ observation of intercellular cell junctions of a single layer by transmission electron microscopy according to claim 1, wherein: the sterilized Epon-812 epoxy resin sheet in the step (1) adopts a sterilization mode of double-sided irradiation by using an ultraviolet lamp.
3. The method for in situ observation of intercellular cell junctions of a single layer by transmission electron microscopy according to claim 1, wherein: the culture conditions of the cells in the incubator are 37 ℃ and 5% CO2。
4. The method for in situ observation of intercellular cell junctions of a single layer according to claim 3, characterized by the following steps: the cell density of the human pancreatic ductal epithelial cells HPDE6C7 added to the culture medium plate was 1 x 10^ 6/ml/well.
5. The method for in situ observation of intercellular cell junctions of a single layer according to claim 4 by transmission electron microscopy, wherein: the length, the width and the height of the cell dense block in the step (3) a are 3mm multiplied by 0.5 mm.
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| 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 |
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| CN110082380A (en) | 2019-08-02 |
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