CN112903401B - Ultrathin section dyeing device - Google Patents
Ultrathin section dyeing device Download PDFInfo
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- CN112903401B CN112903401B CN202110260995.5A CN202110260995A CN112903401B CN 112903401 B CN112903401 B CN 112903401B CN 202110260995 A CN202110260995 A CN 202110260995A CN 112903401 B CN112903401 B CN 112903401B
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- 238000004043 dyeing Methods 0.000 title claims abstract description 293
- 239000007788 liquid Substances 0.000 claims abstract description 105
- 239000012154 double-distilled water Substances 0.000 claims abstract description 19
- 238000002347 injection Methods 0.000 claims description 52
- 239000007924 injection Substances 0.000 claims description 52
- 238000010186 staining Methods 0.000 claims description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- QNRATNLHPGXHMA-XZHTYLCXSA-N (r)-(6-ethoxyquinolin-4-yl)-[(2s,4s,5r)-5-ethyl-1-azabicyclo[2.2.2]octan-2-yl]methanol;hydrochloride Chemical compound Cl.C([C@H]([C@H](C1)CC)C2)CN1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OCC)C=C21 QNRATNLHPGXHMA-XZHTYLCXSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 229920003023 plastic Polymers 0.000 claims description 6
- 210000002445 nipple Anatomy 0.000 claims description 3
- 239000004033 plastic Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 27
- 239000000975 dye Substances 0.000 description 29
- 239000000523 sample Substances 0.000 description 13
- 239000000243 solution Substances 0.000 description 11
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 239000012472 biological sample Substances 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 239000012487 rinsing solution Substances 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- MFEVGQHCNVXMER-UHFFFAOYSA-L 1,3,2$l^{2}-dioxaplumbetan-4-one Chemical compound [Pb+2].[O-]C([O-])=O MFEVGQHCNVXMER-UHFFFAOYSA-L 0.000 description 2
- 229910000003 Lead carbonate Inorganic materials 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 2
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 239000012932 acetate dye Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000013013 elastic material Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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Classifications
-
- 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
- G01N1/31—Apparatus therefor
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses an ultrathin section dyeing device which comprises a dyeing box cover, a dyeing box body, a section box cover, a section box body, a clamping groove and an injector. The invention provides a safe, simple and convenient high-flux section dyeing device which can prevent sections from being disordered or falling off and lost in the dyeing and rinsing processes, avoid the section from being polluted by contacting with air, effectively control the flow rate of the double distilled water for rinsing the sections, improve the dyeing efficiency and quality of ultrathin sections, effectively control the dyeing liquid amount and reduce the pollution to the environment.
Description
Technical Field
The invention relates to a technology for dyeing a sample, in particular to an ultrathin section dyeing device. The section dyeing device is used for observation of a transmission electron microscope and is applied to research in the fields of medical biology and the like.
Background
The transmission electron microscope technology is applied to the observation of the ultrastructure of biological samples and the like, and plays an important role in medical biology research. According to the traditional preparation method of the transmission electron microscope specimen, after a biological sample is fixed, dehydrated, dried, embedded and ultrathin-sliced, the ultrathin slice is fished up to a carrying net such as a copper net or a nickel net, and then double dyeing of uranyl acetate and lead citrate is carried out on the ultrathin slice. The traditional ultrathin section dyeing method is mostly a drop dyeing method or a pinch dyeing method and the like. Wherein, the drop dyeing method comprises the following steps: dropping the dye liquid on a wax sheet or a sealing film, clamping a carrying net with an ultrathin section of the biological sample by using tweezers, covering the sample surface facing the dye liquid drop to float on the liquid drop, covering the liquid drop on a vessel to seal the liquid drop for dyeing, clamping the carrying net by using the tweezers after dyeing, and rinsing by using double distilled water. The disadvantages of this method are: 1. in the drop dyeing method, each liquid drop is covered with a sample carrying net, when a plurality of sample slices are required to be dyed, the time for clamping the carrying nets one by one to cover the liquid drops is longer, the time for exposing the dye liquor in the air is long, and the dye liquor is easy to combine with carbon dioxide in the air to form lead carbonate particle precipitation to cause pollution and influence the subsequent slice observation; 2. in the dyeing process of the drop dyeing method, a vessel covered with dyeing liquid drops needs to be opened for many times, so that the probability of pollution caused by lead carbonate particle precipitation formed by combining the dyeing liquid with carbon dioxide in the air is increased, and the subsequent slice observation is influenced; 3. the drop dyeing method can only carry out dyeing and rinsing on single samples one by one, the slicing is easy to be disordered when the number of the samples is large, the dyeing time of each sample is difficult to control the consistency, the time consumption is long, and the dyeing efficiency is low; 4. the drop dyeing method needs to clamp the ultrathin section carrying net for dyeing and rinsing by using tweezers for multiple times, and the carrying net or a sample section on the carrying net is easily damaged in the clamping process. Another conventional dyeing method commonly used is the clip dyeing method, which comprises the steps of: vertically clamping the carrying net with the ultrathin section on a paraffin dyeing plate, or clamping the carrying net with the ultrathin section in a section hole on a dyeing plate or a rubber tube made of elastic materials such as a silica gel plate, a rubber plate and the like, dropwise adding a dye solution to the sample surface of the ultrathin section carrying net, covering the dyeing plate or the tube with a utensil for dyeing, clamping the dyeing plate with a pair of tweezers for double distilled water rinsing after dyeing. The disadvantages of this method are: 1. the clamp dyeing method needs to clamp the mesh carrying with the ultrathin section into a dyeing plate, so that the mesh carrying is easily clamped and damaged in the operation process, the mesh carrying wrinkles influence subsequent dyeing, and the experimental result is influenced; 2. at present, a commercialized dyeing plate is dyed by a clamp dyeing method, the number of samples at one time is about 25, and the dyeing requirement of a disposable high-flux sample is difficult to meet; 3. a few elastic dyeing plates capable of dyeing more than 50 pieces at one time need to be tightly clamped together, cannot be marked independently one by one and are easy to disorder; 4. in the operation process of the clamp dyeing method, a vessel covering the dyeing plate is often opened for multiple times, the dyeing plate clamp is taken out to rinse the sample, and the sample is easy to contact with air to cause pollution and influence the subsequent section observation; 5. the clamp dyeing method is easy to cause the situation that the net falls off in the dyeing and rinsing process due to unstable clamping, and in addition, if the dyeing plate clamped with the net is directly placed under double-distilled water for rinsing, the water flow speed is difficult to effectively control, the net falls off or the ultrathin section of the biological sample attached to the net is broken or falls off and lost easily, so that the subsequent electron microscope observation is influenced.
Disclosure of Invention
The invention provides an ultrathin section dyeing device aiming at the defects in the prior art.
The technical scheme for solving the technical problems is as follows:
an ultrathin section dyeing device consists of a dyeing box cover, a dyeing box body, a section box cover, a section box body, a clamping groove and an injector;
1. structures for various parts
The dyeing box cover and the dyeing box body are rectangular hollow trapezoid bodies, the area of the bottom of the dyeing box cover is larger than that of the top, the area of the bottom of the dyeing box body is smaller than that of the top, and the top of the dyeing box body can be tightly covered by the bottom of the dyeing box cover.
The top left corner of the dyeing box cover is provided with a liquid injection hole of the dyeing box cover, and the diameter of the liquid injection hole is 3mm.
The dyeing box body divide into dyeing box body upper strata, dyeing box body middle level, dyeing box body lower floor, and the height on dyeing box body upper strata is 25mm, and the height of dyeing box body middle level and dyeing box body lower floor is the same, is 3mm, and the bottom length of dyeing box body lower floor is 74.05mm, and the width is 31.05mm.
The short lateral wall of dyeing box body lower floor length lateral wall junction of dyeing box body has the outage, and the hole diameter is 3mm, has drainage hole stopper through flat silica gel strip subsidiary on the continuous length lateral wall, and the hole stopper is solid cylinder, and the diameter is 2.99mm, and the height is the same with the thickness of dyeing box body.
The length of section lid is 74mm, and the width is 53mm, and high 3mm for having the cavity cuboid of section lid notes liquid hole, section box lid draw-in groove, the liquid hole is located the top upper left corner of section lid is annotated to the section lid, and the hole diameter is 3mm, and the unilateral inslot length of section lid draw-in groove is 74mm, and the width is 9mm, highly is 1mm.
The holes of the slicing box cover are round holes, the number of the holes is 5 rows and 20 columns, each column is aligned at equal height, all the columns are arranged in parallel at equal intervals, the total number of the holes is 100, and the diameter of each hole is 2.5mm.
The liquid hole is annotated to the section box body for having the section box body, the section hole, section hole label, the T type body that section box body wing and reverse side bottom have section hole bottom hole, length is 74mm, positive width is 51mm, reverse side bottom width is 31mm, whole height is 5mm, wherein, the section box body is annotated the hole and is located the positive upper left corner of section box body, hole diameter is 3mm, section hole label is located the positive left side and the downside of section box body respectively, the width of unilateral section box body wing is 10mm, highly be 0.95mm, holistic length is 74mm behind the section box body suit income section lid, the width is 53mm, highly be 6mm.
The slicing holes are rhombic holes with partially closed bottoms, the number of the rows is 5, the rows is 20, every other row is aligned in the same height, all rows are arranged in parallel at equal intervals, the total number of the holes is 100, the diagonal length of the holes is 3.05mm, and the depth of the holes is 4mm.
The bottom holes of the slicing holes are round holes which are positioned at the bottom of the reverse side of the slicing box body, the bottom through holes of the rhombic slicing holes are arranged in 20 rows in total, every two rows are aligned at equal heights, all the rows are arranged in parallel at equal intervals, the total number of the holes is 100, and the diameter of each hole is 2.5mm.
The draw-in groove has four side arms altogether, is located two short lateral wall bottoms on dyeing box body upper strata and up 3mm department, from short lateral wall toward the unsettled extension in the dyeing box body, and each side arm inslot length is 10mm, and the width is 10mm, highly is 3mm.
The injector is a universal injector, the specification is 2 ml-10 ml, and the diameter of the nipple is 3mm.
The dyeing box cover hole is provided with a cross-shaped notch, and the notch is closed in a non-pressure state.
2. Connection relation of each component
The slicing box cover clamping groove is sleeved outside the slicing box body wing, the slicing box cover and the slicing box body form a tightly connected whole, and the whole is tightly sleeved in the clamping groove; the upper layer of the dyeing box body and the middle layer of the dyeing box body are vertically pressed downwards and folded downwards to form the sequential sleeving of the upper layer of the dyeing box body, the middle layer of the dyeing box body and the lower layer of the dyeing box body from outside to inside, so that the bottom of the slicing box is attached and horizontally arranged at the bottom of the lower layer of the dyeing box body, and the lower part of the T-shaped slicing box body is tightly wrapped in the lower layer of the dyeing box body; when the dyeing box body is folded, the liquid injection hole of the dyeing box cover, the liquid injection hole of the slicing box cover and the liquid injection hole of the slicing box body are vertically communicated in parallel; before dyeing, the dyeing box cover is sleeved on the dyeing box body and tightly connected with the dyeing box body, the liquid discharge hole plug is plugged into the liquid discharge hole, and a closed environment is formed in the whole dyeing box; sucking air in the dyeing box through a liquid injection hole or a liquid discharge hole of the dyeing box cover by using a syringe; during dyeing, a syringe is used for injecting dye liquor into the slice box through the liquid injection hole of the dyeing box cover, the liquid injection hole of the slice box cover and the liquid injection hole of the slice box body in sequence for dyeing; after dyeing, the upper layer of the dyeing box body and the middle layer of the dyeing box body extend upwards to restore the original positions by lifting and pulling the upper layer of the dyeing box body, the liquid discharge hole plug is pulled out to open the liquid discharge hole, dye liquid is sucked out and recycled through the liquid discharge hole by using an injector, and double distilled water for rinsing and slicing is discharged subsequently.
The dyeing box cover, the dyeing box body and the clamping groove are made of transparent silica gel with good elasticity, the slicing box cover is made of transparent plastic with stable acid and alkali resistance, and the slicing box body is made of plastic with stable acid and alkali resistance.
The slice hole labels respectively use Arabic numerals or Greek letters and English letters, and the rows and the columns of the slice holes are correspondingly marked in sequence.
The bottom of the lower layer of the dyeing box body can be well adhered to a smooth plane and is not easy to slide, and external force is applied to the side face of the bottom of the lower layer of the dyeing box body to lift upwards, so that the lower layer of the dyeing box body is separated from the original firmly adhered smooth plane.
The invention has the advantages that:
1. the invention can simultaneously dye up to 100 samples at one time by using the slice box with the slice box cover hole, the slice hole label and the slice hole bottom hole, meets the requirement of simultaneously dyeing multiple samples, and each slice is independently placed in one slice hole, so that the slice can be prevented from falling out after the slice box cover is sleeved, the corresponding slice hole label can help to distinguish the samples in each hole, the phenomena of disordered slice positions, falling off and loss can not occur in the dyeing and rinsing processes, and the dyeing efficiency is obviously improved.
2. The dyeing method disclosed by the invention has the advantages that the ultrathin section is dyed by using the section box with the section box cover hole, the section hole and the section hole bottom hole, the three holes are vertically communicated, the dye solution and the double distilled water can completely immerse the ultrathin section in the section hole through the communicated holes, only the dye solution and the double distilled water need to be injected and discharged in the dyeing and rinsing processes, a carrier net does not need to be clamped by tweezers, and the condition that the section is easy to damage due to multiple clamping is effectively avoided.
3. According to the invention, the dyeing box cover and the dyeing box body made of transparent silica gel are used for closed dyeing, after air in the dyeing box is sucked by using the injector before dyeing, the box cover and the box body are not required to be separated in the subsequent dyeing and rinsing processes of ultrathin sections, dyeing and double distilled water injection are only required to be carried out through the liquid injection hole in the dyeing box cover, the recovery of the dyeing liquid and the discharge of the double distilled water are carried out through the liquid discharge hole in the dyeing box body, the contact between the sections and the air is effectively avoided, and the probability of pollution caused by the precipitation generated by the combination of the dyeing liquid and carbon dioxide in the air is greatly reduced.
4. According to the invention, the clamping groove made of silica gel with good elasticity is used, so that the slicing box provided with ultrathin sections can be integrally and stably sleeved on the upper layer of the dyeing box body, when ultrathin sections are dyed, the dyeing box body is pressed and folded, the bottom of the slicing box body can be flatly attached to the bottom of the dyeing box body, about 2.5ml of dye solution is injected into the liquid injection hole of the slicing box body from outside to inside through the liquid injection hole of the dyeing box cover and the liquid injection hole of the slicing box cover in sequence by using the injector, the hydrophobicity of the surface of the bottom of the dyeing box can enable the dye solution to enter the slicing holes through the bottom holes of the slicing holes, so that all the slicing holes of the whole slicing box are filled, and the possible impact damage caused by directly injecting the dye solution into the slices can be effectively avoided; lifting after dyeing and drawing the dyeing box body and making it resume, can making the section box unsettled in the dyeing box body, annotate the liquid hole through the dyeing lid and pour into double distilled water and carry out the rinsing when, double distilled water can link up and flow through the dyeing lid and annotate liquid hole, section box lid hole, section hole bottom hole, realizes the gentle rinsing to the ultra-thin section in the section hole, effectively avoids directly washing the damage that causes the section.
5. The dyeing box made of transparent silica gel is used for dyeing, so that the condition of a sample can be easily observed through the box cover; and the bottom of the slicing box body is tightly wrapped in the lower layer of the dyeing box, the volume is fixed, the dyeing liquid amount can be better controlled, the waste of the dyeing liquid is avoided, and the pollution of the heavy metal dyeing liquid to the environment is favorably reduced.
Drawings
FIG. 1 is a schematic view showing the structure of a slide staining apparatus according to the present invention.
In the figure: the dyeing box comprises a dyeing box cover 1, a dyeing box body 2, a slicing box cover 3, a slicing box body 4, a clamping groove 5, an injector 6, a dyeing box cover liquid injection hole 7, a dyeing box body upper layer 8, a dyeing box body middle layer 9, a dyeing box body lower layer 10, a liquid discharge hole 11, a liquid discharge hole plug 12, a slicing box cover liquid injection hole 13, a slicing box cover hole 14, a slicing box cover clamping groove 15, a slicing box body liquid injection hole 16, a slicing hole 17, a slicing hole label 18, a slicing box body wing 19, a slicing hole bottom hole 20 and a cross notch 21.
FIG. 2 is a transmission electron microscope image obtained after staining using a section staining apparatus according to an embodiment of the present invention.
Detailed Description
The invention is described in further detail below with reference to the figures and specific embodiments.
As shown in figure 1, the ultrathin section dyeing device comprises a dyeing box cover 1, a dyeing box body 2, a section box cover 3, a section box body 4, a clamping groove 5 and an injector 6.
1. Structures for various parts
Dyeing lid 1 and dyeing box body 2 are the hollow prismatoid that top and bottom all are rectangle, and the bottom area of dyeing lid 1 is greater than the top area, and the bottom area of dyeing box body 2 is less than the top area, and the top of dyeing box body 2 can closely be covered to the bottom of dyeing lid 1.
The upper left corner of the top of the dyeing box cover 1 is provided with a liquid injection hole 7 of the dyeing box cover, and the diameter of the hole is 3mm.
Dyeing box body 2 divide into dyeing box body upper strata 8, dyeing box body middle level 9, dyeing box body lower floor 10, and the height of dyeing box body upper strata 8 is 25mm, and the height of dyeing box body middle level 9 and dyeing box body lower floor 10 is the same, is 3mm, and the bottom length of dyeing box body lower floor 10 is 74.05mm, and the width is 31.05mm.
Have outage 11 on the short lateral wall of dyeing box body lower floor 10 long and short lateral wall junction of dyeing box body 2, the hole diameter is 3mm, has drainage hole stopper 12 through flat silica gel strip incidentally on the continuous long lateral wall, and the hole stopper is solid cylinder, and the diameter is 2.99mm, and the height is the same with the thickness of dyeing box body lower floor 10.
The length of section lid 3 is 74mm, and the width is 53mm, and high 3mm for having the cavity cuboid of section lid notes liquid hole 13, section lid hole 14, section lid draw-in groove 15, section lid notes liquid hole 13 is located the top upper left corner of section lid 3, and the hole diameter is 3mm, and the unilateral inslot length of section lid draw-in groove 15 is 74mm, and the width is 9mm, highly is 1mm.
The slicing box cover holes 14 are round holes, the number of the holes is 5 rows and 20 columns, every other column is aligned in the same height, all the columns are arranged in parallel at equal intervals, the total number of the holes is 100, and the diameter of each hole is 2.5mm.
Liquid injection hole 16 is annotated to section box body 4 for having the section box body, section hole 17, section hole label 18, the T type body that section box body wing 19 and reverse side bottom have section hole bottom hole 20, length is 74mm, the front width is 51mm, reverse side bottom width is 31mm, whole height is 5mm, wherein, section box body annotates the positive upper left corner that liquid hole 16 is located section box body 4, the hole diameter is 3mm, section hole label 18 is located the positive left side and the downside of section box body 4 respectively, the width of unilateral section box body wing 19 is 10mm, highly be 0.95mm, holistic length is 74mm after section box body 4 suit income section lid 3, the width is 53mm, highly be 6mm.
The slicing holes 17 are rhombic holes with partially closed bottoms, the number of the rows is 5, the rows are 20, every other row is aligned in the same height, all rows are arranged in parallel at equal intervals, the total number of the holes is 100, the diagonal length of the holes is 3.05mm, and the depth of the holes is 4mm.
The slicing hole bottom holes 20 are round holes which are positioned at the bottom of the reverse side of the slicing box body 4 and are bottom penetrating holes of the rhombic slicing holes 17, 5 rows and 20 columns are formed, every other column is aligned at the same height, all columns are arranged in parallel at equal intervals, total 100 holes are formed, and the diameter of each hole is 2.5mm.
Draw-in groove 5 has four side arms altogether, is located dyeing box body 8 two short lateral wall bottoms 3mm department up, from short lateral wall toward dyeing box body 2 interior unsettled extension, and each side arm inslot length is 10mm, and the width is 10mm, highly is 3mm.
The injector 6 is a universal injector, the specification is 2 ml-10 ml, and the diameter of the nipple is 3mm;
the staining box cover hole 7 is provided with a cross-shaped notch 21 which is closed in a non-pressure state.
Connection relationship of each component
The slicing box cover clamping groove 15 is sleeved outside the slicing box body wing 19, the slicing box cover 3 and the slicing box body 4 form a tightly connected whole, and the whole is tightly sleeved in the clamping groove 5; the upper layer 8 of the dyeing box body and the middle layer 9 of the dyeing box body are vertically pressed downwards and folded downwards to form the upper layer 8 of the dyeing box body, the middle layer 9 of the dyeing box body and the lower layer 10 of the dyeing box body which are sequentially sleeved from outside to inside, so that the bottom of the slicing box is attached to and flatly placed at the bottom of the lower layer 10 of the dyeing box body, and the lower part of the T-shaped slicing box body 4 is tightly wrapped in the lower layer 10 of the dyeing box body; when the dyeing box body 2 is folded, the liquid injection hole 7 of the dyeing box cover, the liquid injection hole 13 of the slicing box cover and the liquid injection hole 16 of the slicing box body are vertically communicated in parallel; before dyeing, the dyeing box cover 1 is sleeved on the dyeing box body 2 and tightly connected, the liquid discharge hole plug 12 is plugged into the liquid discharge hole 11, and a closed environment is formed in the whole dyeing box; air in the dyeing box is sucked through a liquid injection hole 7 or a liquid discharge hole 11 of the dyeing box cover by using an injector 6; during dyeing, a dye solution is injected into the slice box through a dyeing box cover injection hole 7, a slice box cover injection hole 13 and a slice box body injection hole 16 in sequence by using an injector 6 for dyeing; after dyeing, the upper layer 8 of the dyeing box body is lifted and pulled, the upper layer 8 of the dyeing box body and the middle layer 9 of the dyeing box body extend upwards to restore the original positions, the drainage hole plug 12 is pulled out to open the drainage hole 11, the dye liquor is sucked out and recovered through the drainage hole 11 by using the injector 6, and then the double distilled water for rinsing the slices is discharged.
The dyeing box cover 1, the dyeing box body 2 and the clamping groove 5 are made of transparent silica gel with good elasticity, the slicing box cover 3 is made of transparent plastic with stable acid and alkali resistance, and the slicing box body 4 is made of plastic with stable acid and alkali resistance
The above-mentioned chip hole labels 18 are respectively marked with the row and column of the chip holes 17 in order using arabic numerals, greek letters, or the like, and english letters.
The bottom of the lower layer 10 of the dyeing box body can be well adhered to a smooth plane and is not easy to slide, and the lower layer 10 of the color box body is separated from the original firmly adhered smooth plane by applying external force to the side surface of the bottom of the lower layer 10 of the dyeing box body to lift upwards.
The method for dyeing the sample by using the ultrathin section dyeing device comprises the following steps:
1. preparing: ultrathin sections to be dyed are sequentially placed into the section holes 17 of the section box body 4 one by one, the section box body 4 is sleeved into the section box cover clamping groove 15 in parallel through the section box body wings 19, the section box cover 3 is tightly sleeved outside the section box body 4 to form a whole section box, then the section box body is tightly sleeved in the clamping groove 5, the upper layer 8 of the dyeing box body 2 is vertically pressed downwards, the middle layer 9 of the dyeing box body is folded downwards, the upper layer 8 of the dyeing box body, the middle layer 9 of the dyeing box body and the lower layer 10 of the dyeing box body are sequentially sleeved from outside to inside, the bottom of the section box tightly sleeved in the clamping groove 5 is attached to the bottom of the lower layer 10 of the dyeing box body 2, and the lower part of the T-shaped section box body 4 is tightly wrapped in the lower layer 10 of the dyeing box body; when the dyeing box body 2 is folded, the injection hole 7 of the dyeing box cover, the injection hole 13 of the slicing box cover and the injection hole 16 of the slicing box body are communicated vertically and parallelly.
2. The dyeing box cover 1 is sleeved on the dyeing box body 2 to realize tight connection, the liquid discharge hole plug 12 is sleeved in the liquid discharge hole 11, and a closed environment is formed in the whole dyeing box.
3. Air in the closed dyeing box is sucked out from the liquid injection hole 7 of the dyeing box cover by using the syringe 6.
4. About 2.5ml of uranyl acetate dye liquor sequentially passes through a dye box cover liquid injection hole from outside to inside by using an injector 6, the dye liquor is injected into a section box body liquid injection hole through the section box cover liquid injection hole, the hydrophobicity of the surface of the bottom of the dye box can enable the dye liquor to enter the section hole through a section hole bottom hole, so that all section holes of the whole section box are filled, the height of the dye liquor is observed through a transparent dye box cover, the dye liquor is slightly higher than or level to the cover hole of the section box, the ultra-thin sections in the section hole can be completely immersed by the dye liquor, the injector is drawn out, and the standing dyeing is carried out for 15-25 min.
5. Lifting and pulling the upper layer of the dyeing box body, extending and recovering the middle layer of the dyeing box body folded downwards upwards to restore the original positions of the upper layer of the dyeing box body, the middle layer of the dyeing box body and the lower layer of the dyeing box body, suspending the slicing box in the dyeing box body, drawing the liquid drainage hole plug to open the liquid drainage hole, and sucking and recovering the dye liquor through the liquid drainage hole by using an injector.
6. The liquid discharge hole plug is plugged into the liquid discharge hole to seal the liquid discharge hole, double distilled water is slowly injected into the dyeing box along the liquid injection hole of the dyeing box cover by using the injector, the liquid level in the dyeing box is observed to ensure that the section box is completely immersed by the double distilled water, the dyeing box can be gently shaken to further improve the rinsing effect, after rinsing is carried out for 1-5 min, the injector is used for discharging the double distilled water for rinsing the sections, and the step is repeated for at least 3 times.
7. Gently flick the staining box to promote the outflow of the residual rinsing solution, and suck out the residual rinsing solution completely by using an injector.
8. Press the dyeing box body upper 8 of dyeing box body 2 down perpendicularly, suit dyeing box body upper 8, dyeing box body middle level 9, dyeing box body lower floor 10 in proper order from outer to inner once more, make the laminating of the section box bottom of closely suit in draw-in groove 5 flat arrange dyeing box body lower floor 10 bottom in, and then realize that the lower part of T type section box body 4 closely wraps up in dyeing box body lower floor 10.
9. The drainage hole plug 12 is sleeved in the drainage hole 11, and a closed environment is formed in the whole dyeing box.
10. The air in the closed dyeing box is sucked out from the liquid injection hole 7 of the dyeing box cover by using the injector 6
11. And (3) injecting about 2.5ml of lead citrate dye solution into the liquid injection hole of the slice box body from outside to inside through the liquid injection hole of the dyeing box cover and the liquid injection hole of the slice box cover in sequence by using an injector 6, observing the height of the dye solution through the transparent dyeing box cover until the liquid level of the dye solution is slightly higher than or flush with the hole of the slice box cover, ensuring that the dye solution can completely immerse the ultrathin slices in the slice hole, drawing out the injector, and standing and dyeing for 5-10 min.
12. Lifting and pulling the upper layer of the dyeing box body, extending and recovering the middle layer of the dyeing box body folded downwards upwards to restore the original positions of the upper layer of the dyeing box body, the middle layer of the dyeing box body and the lower layer of the dyeing box body, suspending the slicing box in the dyeing box body, drawing the liquid drainage hole plug to open the liquid drainage hole, and sucking and recovering the dye liquor through the liquid drainage hole by using an injector.
13. The liquid discharge hole plug is plugged into the liquid discharge hole to seal the liquid discharge hole, double distilled water is slowly injected into the dyeing box along the liquid injection hole of the dyeing box cover by using the injector, the liquid level in the dyeing box is observed to ensure that the section box is completely immersed by the double distilled water, the dyeing box can be gently shaken to further improve the rinsing effect, after rinsing is carried out for 1-5 min, the injector is used for discharging the double distilled water for rinsing the sections, and the step is repeated for at least 3 times.
14. Gently flick the staining box to promote the outflow of the residual rinsing solution, and suck out the residual rinsing solution completely by using an injector.
15. And (3) opening the staining box and the slice box in sequence, clamping the ultrathin slices one by using tweezers to form slice holes, sucking rinse liquid possibly remaining on the slices by using dust-free filter paper, putting the slices on the dust-free filter paper in a glass culture dish, and drying in an oven at 37 ℃ for later use.
16. The stained sample was observed and imaged by transmission electron microscopy as shown in FIG. 2.
In summary, the above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.
Claims (4)
1. The utility model provides an ultra-thin section dyeing apparatus which characterized in that: the device consists of a staining box cover (1), a staining box body (2), a slicing box cover (3), a slicing box body (4), a clamping groove (5) and an injector (6);
structures for various parts
The top and the bottom of the dyeing box cover (1) and the dyeing box body (2) are rectangular hollow trapezoid bodies, the area of the bottom of the dyeing box cover (1) is larger than that of the top, the area of the bottom of the dyeing box body (2) is smaller than that of the top, and the bottom of the dyeing box cover (1) can tightly cover the top of the dyeing box body (2);
the left upper corner of the top of the dyeing box cover (1) is provided with a dyeing box cover liquid injection hole (7) with the diameter of 3mm;
the dyeing box body (2) is divided into a dyeing box body upper layer (8), a dyeing box body middle layer (9) and a dyeing box body lower layer (10), the height of the dyeing box body upper layer (8) is 25mm, the heights of the dyeing box body middle layer (9) and the dyeing box body lower layer (10) are the same and are 3mm, the bottom length of the dyeing box body lower layer (10) is 74.05mm, and the width of the dyeing box body lower layer (10) is 31.05mm;
the short side wall of the joint of the long side wall and the short side wall of the lower layer (10) of the dyeing box body (2) is provided with a liquid discharge hole (11), the diameter of the hole is 3mm, the long side wall connected with the hole is additionally provided with a liquid discharge hole plug (12) through a flat silica gel strip, the hole plug is a solid cylinder, the diameter of the hole plug is 2.99mm, and the height of the hole plug is the same as the thickness of the lower layer (10) of the dyeing box body;
the length of the slicing box cover (3) is 74mm, the width is 53mm, the height is 3mm, the slicing box cover is a hollow cuboid with a slicing box cover liquid injection hole (13), a slicing box cover hole (14) and a slicing box cover clamping groove (15), the slicing box cover liquid injection hole (13) is positioned at the upper left corner of the top of the slicing box cover (3), the hole diameter is 3mm, the length of the single-side groove of the slicing box cover clamping groove (15) is 74mm, the width is 9mm, and the height is 1mm;
the slicing box cover holes (14) are round holes, the number of the holes is 5 rows and 20 columns, every two columns are aligned at the same height, all the columns are arranged in parallel at equal intervals, the total number of the holes is 100, and the diameter of each hole is 2.5mm;
the slicing box body (4) is a T-shaped body with a slicing box body liquid injection hole (16), a slicing hole (17), a slicing Kong Biaoqian (18), a slicing box body wing (19) and a slicing hole bottom hole (20) in the bottom of the back side, the length is 74mm, the front width is 51mm, the back side bottom width is 31mm, and the whole height is 5mm, wherein the slicing box body liquid injection hole (16) is located in the upper left corner of the front side of the slicing box body (4), the hole diameter is 3mm, the slicing Kong Biaoqian (18) is respectively located on the left side and the lower side of the front side of the slicing box body (4), the width of the single-side slicing box body wing (19) is 10mm, the height is 0.95mm, the slicing box body (4) is sleeved with the slicing box cover (3), the whole length is 74mm, the width is 53mm, and the height is 6mm;
the slicing holes (17) are rhombic holes with partially closed bottoms, the number of the rows is 5, the number of the rows is 20, every other row is aligned in the same height, all rows are arranged in parallel at equal intervals, the total number of the holes is 100, the diagonal length of each hole is 3.05mm, and the depth of each hole is 4mm;
the slicing hole bottom holes (20) are round holes which are positioned at the bottom of the reverse side of the slicing box body (4), are bottom through holes of rhombic slicing holes (17), and are 20 rows in 5 rows, every two rows are aligned at equal height, all rows are arranged in parallel at equal intervals, the total number of the holes is 100, and the diameter of each hole is 2.5mm;
the clamping grooves (5) are provided with four side arms in total, are positioned at the positions 3mm above the bottoms of two short side walls of the upper layer (8) of the dyeing box body, and are suspended and extended from the short side walls into the dyeing box body (2), and each side arm is 10mm in length, 10mm in width and 3mm in height;
the injector (6) is a universal injector, the specification is 2 ml-10 ml, and the diameter of a nipple is 3mm;
the liquid injection hole (7) of the dyeing box cover is provided with a cross cut (21), and the cut is closed in a non-pressure state;
connection relationship of each component
The slicing box cover clamping groove (15) is sleeved outside the slicing box body wing (19), the slicing box cover (3) and the slicing box body (4) form a tightly connected whole, and the whole is tightly sleeved in the clamping groove (5); the upper dyeing box body layer (8) and the middle dyeing box body layer (9) are vertically pressed downwards and are folded downwards to form the upper dyeing box body layer (8), the middle dyeing box body layer (9) and the lower dyeing box body layer (10) which are sequentially sleeved from outside to inside, so that the bottom of the slicing box is attached to the bottom of the lower dyeing box body layer (10), and the lower part of the T-shaped slicing box body (4) is tightly wrapped in the lower dyeing box body layer (10); when the staining box body (2) is folded, the liquid injection hole (7) of the staining box cover, the liquid injection hole (13) of the slicing box cover and the liquid injection hole (16) of the slicing box body are vertically communicated in parallel; before dyeing, the dyeing box cover (1) is sleeved on the dyeing box body (2) and tightly connected, the liquid drainage hole plug (12) is plugged into the liquid drainage hole (11), and a closed environment is formed in the whole dyeing box; sucking air in the dyeing box through a liquid injection hole (7) or a liquid discharge hole (11) of the dyeing box cover by using a syringe (6); during dyeing, dye liquor is injected into the slice box through a dyeing box cover injection hole (7), a slice box cover injection hole (13) and a slice box body injection hole (16) in sequence by using an injector (6) for dyeing; after dyeing, the upper layer (8) of the dyeing box body and the middle layer (9) of the dyeing box body are upwards extended and restored to the original positions by lifting and pulling the upper layer (8) of the dyeing box body, the liquid drainage hole plug (12) is pulled out to open the liquid drainage hole (11), the dye liquid is sucked out and recovered through the liquid drainage hole (11) by using the injector (6), and then double distilled water for rinsing the slices is discharged.
2. The ultra-thin section staining apparatus of claim 1, wherein: dyeing lid (1), dyeing box body (2), draw-in groove (5) are made for the transparent silica gel that elasticity is good, and section lid (3) are made for the acid and alkali resistance stable transparent plastic, and section box body (4) are made for the acid and alkali resistance stable plastic.
3. The ultra-thin section staining apparatus of claim 1, wherein: the sections Kong Biaoqian (18) are respectively marked with Arabic numerals in sequence corresponding to the rows and columns of the section holes (17).
4. The ultra-thin section staining apparatus of claim 1, wherein: the bottom of the lower dyeing box body layer (10) can be well adhered to a smooth plane and is not easy to slide, and external force is applied to the side face of the bottom of the lower dyeing box body layer (10) to lift upwards, so that the lower dyeing box body layer (10) is separated from the original firmly adhered smooth plane.
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| CA2301366A1 (en) * | 1999-04-13 | 2000-10-13 | Kimberly-Clark Worldwide, Inc. | Apparatus for forming a fibrous mat with improved edge strength |
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| US6735531B2 (en) * | 1996-10-07 | 2004-05-11 | Lab Vision Corporation | Method and apparatus for automatic tissue staining |
| AU2003901871A0 (en) * | 2003-03-31 | 2003-05-08 | Vision Biosystems Limited | A method and apparatus for fluid dispensation, preparation and dilation |
| US7888110B2 (en) * | 2003-06-26 | 2011-02-15 | Seng Enterprises Ltd. | Pico liter well holding device and method of making the same |
| US7521021B2 (en) * | 2003-11-26 | 2009-04-21 | Leica Biosvstems St. Louis Llc | System for in situ processing of a tissue specimen |
| US8518348B2 (en) * | 2003-11-26 | 2013-08-27 | Leica Biosystems Richmond, Inc. | Histological specimen retaining device |
| CN201628650U (en) * | 2010-04-09 | 2010-11-10 | 李学慧 | Portable multipurpose constant temperature dyeing box |
| CN201974327U (en) * | 2011-02-11 | 2011-09-14 | 天津市宝坻区人民医院 | Staining rack for smear and tissue section |
| JP5996550B2 (en) * | 2011-11-24 | 2016-09-21 | オリンパス株式会社 | Cell sorting device and cell sorting method |
| CN203811445U (en) * | 2014-04-29 | 2014-09-03 | 侯文丽 | Disposable common-staining and immunostaining set for transmission electron microscope |
| CN105181425A (en) * | 2015-09-22 | 2015-12-23 | 浙江万里学院 | Cell negative staining plate |
| CN210119387U (en) * | 2019-03-11 | 2020-02-28 | 华中科技大学苏州脑空间信息研究院 | Convenient dyeing box capable of uniformly dyeing samples in batches |
| CN214952525U (en) * | 2021-05-26 | 2021-11-30 | 宁夏医科大学总医院 | Cell climbing film light-resistant dyeing incubation box |
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| CA2301366A1 (en) * | 1999-04-13 | 2000-10-13 | Kimberly-Clark Worldwide, Inc. | Apparatus for forming a fibrous mat with improved edge strength |
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