CN110553888A - Method for rapid tabletting of oocyte after immunofluorescence staining - Google Patents
Method for rapid tabletting of oocyte after immunofluorescence staining Download PDFInfo
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- CN110553888A CN110553888A CN201910875138.9A CN201910875138A CN110553888A CN 110553888 A CN110553888 A CN 110553888A CN 201910875138 A CN201910875138 A CN 201910875138A CN 110553888 A CN110553888 A CN 110553888A
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- glass slide
- oocyte
- adhesive tape
- edge
- slide
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- 210000000287 oocyte Anatomy 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000003125 immunofluorescent labeling Methods 0.000 title claims abstract description 8
- 239000011521 glass Substances 0.000 claims abstract description 35
- 239000002390 adhesive tape Substances 0.000 claims abstract description 25
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000011248 coating agent Substances 0.000 claims abstract description 3
- 238000000576 coating method Methods 0.000 claims abstract description 3
- 238000003825 pressing Methods 0.000 claims abstract description 3
- 238000007789 sealing Methods 0.000 claims abstract description 3
- 210000002268 wool Anatomy 0.000 claims abstract description 3
- 239000004166 Lanolin Substances 0.000 claims description 4
- 229940039717 lanolin Drugs 0.000 claims description 4
- 235000019388 lanolin Nutrition 0.000 claims description 4
- 238000010791 quenching Methods 0.000 abstract description 6
- 230000000171 quenching effect Effects 0.000 abstract description 6
- 238000010186 staining Methods 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 abstract description 2
- 230000009456 molecular mechanism Effects 0.000 abstract description 2
- 230000008182 oocyte development Effects 0.000 abstract description 2
- 239000006059 cover glass Substances 0.000 description 6
- 239000008188 pellet Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 108010077544 Chromatin Proteins 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 102000002151 Microfilament Proteins Human genes 0.000 description 2
- 108010040897 Microfilament Proteins Proteins 0.000 description 2
- 210000003483 chromatin Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000116 DAPI staining Methods 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 239000011505 plaster Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
A method for rapid tabletting of oocyte after immunofluorescence staining belongs to the technical field of cell biology. In order to reduce the problem of fluorescence quenching during observation after oocyte staining, the invention provides a method for quickly tabletting after oocyte immunofluorescence staining, which comprises the following steps: taking a glass slide, respectively sticking a small piece of transparent adhesive tape on the upper side and the lower side of the middle position of the glass slide close to the edge, then placing the dyed oocyte and the sealing solution containing DAPI at the middle position of the two adhesive tapes, coating wool fat with the volume of not more than 10 mu L on the four corners of the glass slide, and then pressing the glass slide on the glass slide; the adhesive tape has a width of 0.2-0.3cm, a length of 1.0-1.5cm, and a sticking thickness of 37-78 μm. The invention reduces the risk of fluorescence quenching and provides a new technical support for researching the molecular mechanism of oocyte development.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a method for rapid tabletting of oocyte after immunofluorescence staining.
Background
Oocyte fluorescent staining experiments often involve staining with various antibodies, and often a low-temperature long-time incubation method is adopted to reduce non-specific binding, which already increases the possibility of fluorescent quenching, while the last-step tabletting often needs to be observed under a microscope to ensure that the tabletting degree is good, and the possibility of fluorescent quenching is greatly increased.
Disclosure of Invention
In order to reduce the problem of fluorescence quenching during observation after oocyte staining, the invention provides a method for quickly tabletting after oocyte immunofluorescence staining, which comprises the following steps: taking a glass slide, respectively sticking a small piece of transparent adhesive tape on the upper side and the lower side of the middle position of the glass slide close to the edge, then placing the dyed oocyte and the sealing solution containing DAPI at the middle position of the two adhesive tapes, coating wool fat with the volume of not more than 10 mu L on the four corners of the glass slide, and then pressing the glass slide on the glass slide; the width of the adhesive tape is 0.2-0.3cm, the length of the adhesive tape is 1.0-1.5cm, and the pasting thickness is 37-78 μm; one end of the adhesive tape close to the wide edge of the glass slide is 1.0-1.4cm away from the wide edge of the glass slide; the distance between the long edge of the adhesive tape close to the edge of the glass slide and the edge of the glass slide is less than 0.2 cm.
Preferably, the adhesive tape is 0.3cm wide and 1.2cm long.
Preferably, one end of the adhesive tape close to the wide edge of the glass slide is 1.1cm away from the wide edge of the glass slide.
Preferably, the slide format is 25mm by 75 mm; the cover slip specification is 25 mm.
Preferably, the lanolin is applied in an amount of 5 μ L.
Advantageous effects
The invention uses simple and easily obtained raw materials to rapidly and simply carry out tabletting, reduces the risk of fluorescence quenching, and provides a new technical support for researching the molecular mechanism of oocyte development.
Drawings
FIG. 1 schematic illustration of oocyte pellet preparation; wherein 1 is a glass slide, 2 is two pieces of adhesive tapes pasted on the glass slide, 3 is a stained oocyte, and 4 is a cover glass;
FIG. 2 diagram of oocyte pellet;
FIG. 3 shows the result of fluorescence microscopy of oocytes, wherein red is an antibody linked to TRITC, green is an antibody linked to FITC, blue is the DAPI staining effect, a is a combined graph, b is a chromatin staining graph, c is a membrane protein staining graph, and d is a microfilament protein staining graph.
Detailed Description
Example 1 method for rapid pelleting of oocytes after immunofluorescent staining.
This example describes a method for rapid preparation of a pellet, using as an example a stained zona pellucida-removed porcine oocyte.
Taking a clean glass slide, wherein the specification is 25mm x 75mm, respectively sticking a small piece of transparent adhesive tape 1 layer at the upper side and the lower side of the middle position close to the edge, the thickness is about 38 mu m, the width of the stuck adhesive tape is 0.3cm, the length of the stuck adhesive tape is 1.2cm, one end of each adhesive tape close to the wide edge of the glass slide is 1.1cm away from the wide edge of the glass slide, and the distance between the long edge of the adhesive tape close to the edge of the glass slide and the edge of the glass slide is less than 0.2cm, as shown in figure 1; the cover glass used in this example is 25mm by 25mm, the stained porcine oocytes and the commercial mounting solution containing DAPI are placed at the center of two adhesive tapes, 5 μ L of lanolin is coated at the four corners of the cover glass to assist in adhering and supporting the cover glass, then the cover glass is pressed on the slide glass, the distance between the cover glass and the edge of the slide glass is 0.5cm, and the detection can be performed by curing the mounting solution in a cassette.
Example 2. example 1 was repeated, differing from example 1 in that the application amount of lanolin in this example was 10 μ L.
Example 3. example 1 was repeated, except that the adhesive plaster of this example was 0.2cm wide and 1.5cm long.
Comparative example 1. example 1 was repeated, except that no adhesive tape was attached to the slide glass in this comparative example, unlike example 1.
Taking example 1 as an example, the porcine oocytes were observed by a confocal laser microscope, and as a result, chromatin showing blue fluorescence, membrane protein showing ring-like green fluorescence, and microfilament protein showing ring-like red fluorescence were clearly seen using the pellet prepared by the method of the present invention. Whereas red or green fluorescence was observed over the entire area in the tabletting method of comparative example 1.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. a method for rapid tabletting after immunofluorescence staining of oocyte is characterized by comprising the following steps: taking a glass slide, respectively sticking a small piece of transparent adhesive tape on the upper side and the lower side of the middle position of the glass slide close to the edge, then placing the dyed oocyte and the sealing solution containing DAPI at the middle position of the two adhesive tapes, coating wool fat with the volume of not more than 10 mu L on the four corners of the glass slide, and then pressing the glass slide on the glass slide; the width of the adhesive tape is 0.2-0.3cm, the length of the adhesive tape is 1.0-1.5cm, and the pasting thickness is 37-78 μm; one end of the adhesive tape close to the wide edge of the glass slide is 1.0-1.4cm away from the wide edge of the glass slide; the distance between the long edge of the adhesive tape close to the edge of the glass slide and the edge of the glass slide is less than 0.2 cm.
2. The method of claim 1, wherein the blanket is 0.3cm wide and 1.2cm long.
3. The method of claim 1, wherein an end of the tape proximate the broad side of the slide is 1.1cm from the broad side of the slide.
4. The method of claim 1, wherein the slide format is 25mm by 75 mm; the cover slip specification is 25 mm.
5. The method of claim 1, wherein the lanolin is applied in an amount of 5 μ L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910875138.9A CN110553888B (en) | 2019-09-16 | 2019-09-16 | Method for rapid tabletting of oocyte after immunofluorescence staining |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910875138.9A CN110553888B (en) | 2019-09-16 | 2019-09-16 | Method for rapid tabletting of oocyte after immunofluorescence staining |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110553888A true CN110553888A (en) | 2019-12-10 |
| CN110553888B CN110553888B (en) | 2022-07-12 |
Family
ID=68740451
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910875138.9A Active CN110553888B (en) | 2019-09-16 | 2019-09-16 | Method for rapid tabletting of oocyte after immunofluorescence staining |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110553888B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4190472A (en) * | 1977-09-28 | 1980-02-26 | Alex Slonicki | Automated system for the application of coverglasses on histological and cytological slides |
| CN101650310A (en) * | 2009-09-17 | 2010-02-17 | 天津师范大学 | Immunofluorescence method for rapidly detecting nucleolus protein positioning under the stress of heavy metal |
| CN201785336U (en) * | 2009-08-03 | 2011-04-06 | 四川大学华西第二医院 | In-situ-hybridization one-step mounting tape |
| CN102308005A (en) * | 2009-01-26 | 2012-01-04 | 泰希斯有限公司 | Functionalized microfluidic device for immunofluorescence |
| CN203930200U (en) * | 2014-07-14 | 2014-11-05 | 重庆科技学院 | Observation by microscope medical treatment count slice |
| CN104266973A (en) * | 2014-10-13 | 2015-01-07 | 中国科学技术大学 | Permanent liquid-state microsphere sample chamber |
| WO2017062646A1 (en) * | 2015-10-07 | 2017-04-13 | Clearbridge Biophotonics Pte Ltd. | Integrated visual morphology and cell protein expression using resonance-light scattering |
| BR102016009754A2 (en) * | 2016-04-29 | 2017-11-07 | Universidade Federal De São Paulo - Unifesp | METHOD OF DETECTION OF PROPER INTAKE OF INHIBITING MEDICATION OF THE INOSINE ENZINE DEHYDROGENASE MONOPHOSPHATE II, USE OF THE METHOD AND KIT FOR EXECUTING THE METHOD |
| CN108254237A (en) * | 2017-12-12 | 2018-07-06 | 浙江海洋大学 | A kind of live cell fluorescent colouring method |
-
2019
- 2019-09-16 CN CN201910875138.9A patent/CN110553888B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4190472A (en) * | 1977-09-28 | 1980-02-26 | Alex Slonicki | Automated system for the application of coverglasses on histological and cytological slides |
| CN102308005A (en) * | 2009-01-26 | 2012-01-04 | 泰希斯有限公司 | Functionalized microfluidic device for immunofluorescence |
| CN201785336U (en) * | 2009-08-03 | 2011-04-06 | 四川大学华西第二医院 | In-situ-hybridization one-step mounting tape |
| CN101650310A (en) * | 2009-09-17 | 2010-02-17 | 天津师范大学 | Immunofluorescence method for rapidly detecting nucleolus protein positioning under the stress of heavy metal |
| CN203930200U (en) * | 2014-07-14 | 2014-11-05 | 重庆科技学院 | Observation by microscope medical treatment count slice |
| CN104266973A (en) * | 2014-10-13 | 2015-01-07 | 中国科学技术大学 | Permanent liquid-state microsphere sample chamber |
| WO2017062646A1 (en) * | 2015-10-07 | 2017-04-13 | Clearbridge Biophotonics Pte Ltd. | Integrated visual morphology and cell protein expression using resonance-light scattering |
| BR102016009754A2 (en) * | 2016-04-29 | 2017-11-07 | Universidade Federal De São Paulo - Unifesp | METHOD OF DETECTION OF PROPER INTAKE OF INHIBITING MEDICATION OF THE INOSINE ENZINE DEHYDROGENASE MONOPHOSPHATE II, USE OF THE METHOD AND KIT FOR EXECUTING THE METHOD |
| CN108254237A (en) * | 2017-12-12 | 2018-07-06 | 浙江海洋大学 | A kind of live cell fluorescent colouring method |
Non-Patent Citations (2)
| Title |
|---|
| MARCIE TOMBLYN ET AL.: "Guidelines for Preventing Infectious Complications among HCT Recipients", 《BIOL BLOOD MARROW TRANSPLANT 15》 * |
| 杨丹等: "Filipin Ⅲ对肺泡巨噬细胞caveolin-1的影响", 《湖南师范大学学报(医学版)》 * |
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| Publication number | Publication date |
|---|---|
| CN110553888B (en) | 2022-07-12 |
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