CN105784991A - Device and method for repeated immunostaining of same tissue section - Google Patents
Device and method for repeated immunostaining of same tissue section Download PDFInfo
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Abstract
The invention discloses a device and method for repeated immunostaining of the same tissue section in order to realize repeated immunostaining of the same tissue section and save tissue samples and meet diagnosis needs, including a tissue section bearing device and an application method thereof. The tissue section bearing device comprises a glass slide with a groove and a cover glass. The tissue section bearing device is adopted for repeated staining of sequentially circulating staining, photographing, cleaning, staining again, photographing again and cleaning again of the same tissue section; and through comparative analysis of pictures taken after the repeated staining, the antigen expression profile of each cell on the tissue section can be obtained.
Description
Technical field
The present invention relates to medical biotechnology experimental provision and method, particularly relating to a kind of apparatus and method for same tissue slice being carried out repeatedly immunostaining.
Background technology
Immunostaining is the principle utilizing antigen to be combined with antibody specificity, the expression of detection specific protein (antigen), it is find one of body detection method that changes of function is the most frequently used under physiology or pathological state, is most commonly used that immunohistochemical staining and immunofluorescence dyeing.Conventional method is that a tissue slice carries out the colouring method of a kind of antigenic mark, and the expression that will study plurality of antigens then needs multiple tissue slices.If minimal amount of tissue specimen (such as cytological samples obtained by fine-needle aspiration) carries out the research of plurality of antigens expression, routine immunization colouring method is owing to being difficult to by specimen quantitative limitation.Therefore, it is necessary to develop a kind of apparatus and method that same tissue slice can be carried out repeatedly immunostaining, to save tissue specimen, meet diagnosis needs.
Summary of the invention
The technical problem to be solved is to be difficult to that minimal amount of tissue specimen carries out plurality of antigens for prior art to express situation research, it is provided that a kind of apparatus and method for same tissue slice carries out repeatedly immunostaining.
This invention address that above-mentioned technical problem the technical scheme is that same tissue slice carries out repeatedly immunostaining adopts tissue slice bogey and repeatedly staining.
Described tissue slice bogey, including band groove microscope slide and coverslip two parts, described being made up of the flint glass of high transparent with groove microscope slide, there are at least one groove in central authorities, and the shape of groove is preferable over cube, groove-bottom is smooth, groove-bottom thickness is (1.0 ± 0.2mm), and depth of groove is 0.5mm to 3mm, according to the described coverslip size intending coupling, the level that is fabricated to by groove puts into coverslip, and makes have gap wide for about 1mm to be advisable between coverslip and groove surrounding sidewall.Described coverslip is commercially available conventional coverslip, and thickness is 0.13-0.17mm, has multiple different size, is made up of high-transparent glass.Groove with groove microscope slide needs to adopt method conventional at present, increases the adhesiveness of tissue and microscope slide groove-bottom as smeared poly-D-lysine etc., to avoid tissue slice to depart from microscope slide.Tissue slice is placed in the groove inner bottom part central authorities with groove microscope slide, and coverslip is placed in above tissue slice.
Described in execution repeatedly before staining, prepare before first carrying out the dyeing of routine, including the section (thickness is 4 ~ 6 μm) of paraffin-embedded tissue, drift sheet, tissue slice is placed in the bottom portion of groove central authorities with groove microscope slide, a series of conventional steps (frozen section can dye after then cutting into slices) such as roasting sheet, dewaxing, aquation, antigen retrieval, coverslip is placed in above tissue slice, the dyeing that then tissue slice circulated successively, the repeatedly dyeing taken pictures, clean, dye again, take pictures again, clean again.Described dyeing, namely the gap place between coverslip and the groove walls with groove microscope slide is added reagent on successively, reagent is made to be spread and submergence tissue slice by capillary effect, after reagent affected tissues cuts into slices a period of time, microscope slide is vertically placed, reagent in groove then flows out, and draws and major part removing with absorbent paper.Operate by test kit description, add the first first antibody and corresponding second antibody etc. successively, to developed the color rear (not carrying out haematoxylin redyeing nucleus and mounting), use buffer color development stopping, microscope slide is vertically placed, suck major part residual buffer with absorbent paper, take pictures described in can carrying out under the microscope.Described take pictures, be about to be put under microscope with the tissue slice bogey of tissue slice, use the integral photograph (amplifying 200 ~ 400 times) after image acquisition device tissue section strain.Described taken pictures after, carry out described cleaning, namely reagent is added color product is fully erased, the phosphate buffer (about 60 DEG C) of available heat removes the first antibody and the second antibody that are attached to tissue slice, difference according to Color Appearance System, dissolves with corresponding reagent and removes the color product being incorporated into tissue slice.After being successfully completed described cleaning, tissue slice recovers the color to dyeing, dyes described in can carrying out again.Described dye again, namely operate to specifications, add the second first antibody and corresponding second antibody etc. successively, after having developed the color, repeat the operation identical with described dyeing, take pictures again described in carrying out, described clean again, then add the third first antibody, the 4th kind of first antibody etc. by above-mentioned circulation, until completing to intend the research of whole antigen presentations of research.Same tissue slice can be carried out 3 times to 20 times dyeing according to described repeatedly staining.Then, as dyeing quality control method, repeat the first first antibody dyeing (purpose be evaluate in repeatedly dyeing course, whether there is antigen Loss, if antigen is lost more than 20%, need to consider to reduce dyeing number of times, namely reduce the dyeing number of times to same tissue slice), after taking pictures, carrying out routine hematoxylin-eosin stains, mounting is also taken pictures.In whole dyeing course, remain that coverslip is covered in tissue slice surface, it is impossible to midway is removed.If whenever tissue slice leaves initial position in whole operating process, then may result in the failure of an experiment.The photo obtained after repeatedly dyeing is compared by computer software, the corresponding antigens express spectra of each cell on tissue slice can be obtained.
The described apparatus and method for same tissue slice being carried out repeatedly immunostaining, it is advantageous that: 1. antigen retrieval complete after all operations step, coverslip keeps covering tissue slice, it is prevented effectively from reagent to impact and cause that tissue slice departs from microscope slide taking pictures after being conducive to each antibody staining to complete；2. effective removing is attached to the first antibody of tissue slice, second antibody and color product, reduces the impact that follow-up antigen is dyeed by the dyeing of previous antigen；3., after the antigen intending research has dyeed, the first antigen of labelling again, as quality control method；4. after completing all immunostainings, carry out routine hematoxylin-eosin stains, make full use of tissue slice, and be more beneficial for analyzing and diagnosing；5. repeatedly dyeing picture is analyzed processing by computer software, can obtain the corresponding antigens express spectra of each cell in tissue slice.
Accompanying drawing explanation
Fig. 1 is the plan view of tissue slice bogey；
Fig. 2 is the longitudinal section view of tissue slice bogey；
In figure: 1 is band groove microscope slide；2 is groove；3 is coverslip；4 is tissue slice.
Specific implementation method
Below in conjunction with accompanying drawing and instantiation, the present invention is described in detail.
As depicted in figs. 1 and 2, tissue slice bogey, including band groove microscope slide 1 and coverslip 3, described band groove microscope slide 1 length is 60mm, and width is 40mm, and except groove 2 is with the thickness of outer portion for 2.5mm, groove 2 bottom thickness is 1.0mm；Described groove 2 length is 42mm, and width is 26mm, and the degree of depth is 1.5mm, and groove inner wall scribbles poly-D-lysine；Described coverslip 3 length is 40mm, and width is 24mm, and thickness is 0.13mm.
Use described tissue slice bogey to realize repeatedly immunostaining, following two kinds of methods can be adopted:
Method one: the paraffin-embedded tissue routine from lymph node puncture specimen is cut into slices (thickness 4 μm), floated sheet, pick up a tissue slice 4 with band groove microscope slide 1 and be positioned over groove 2 central authorities, roasting sheet, dewaxing, aquation, antigen retrieval, closing endogenous catalase routinely, with phosphate buffer (pH value 7.3-7.4 after often having walked, lower same) soak, coverslip 3 is placed in groove 2 and covers tissue slice 4.After following every single stepping completes, erect tissue slice bogey, suck major part remaining reagent with absorbent paper.Monoclonal CD3 antibody working solution 100 microlitre is added into the gap place between coverslip 3 and groove 2, hatch 60 minutes for 37 DEG C, phosphate buffer washes three times, every all over 5 minutes, the second antibody 37 DEG C adding corresponding horseradish peroxidase (HRP) labelling hatches 30 minutes, phosphate buffer is washed three times, every all over 5 minutes, adds AEC (3-amino-9-ethylcarbazole；AEC) colour developing 8 minutes, phosphate buffer washes three times, every all over 5 minutes, tissue slice 4 is scanned under ordinary optical microscope, 1 minute is respectively acted on to wash away red color product successively with dehydrated alcohol and dimethylbenzene, 60 DEG C of phosphate buffer affected tissues are cut into slices 5 minutes, phosphate buffer washes three times, every all over 5 minutes, check under ordinary optical microscope, if finding red color product residual, repeatable 60 DEG C of phosphate buffer affected tissues are cut into slices 5 minutes, and check carries out next dyeing planting antigen after finding tissue slice redfree color product residual；Monoclonal CD4 antibody working solution 100 microlitre is added into the gap place between coverslip and groove, repeat the step after adding monoclonal CD3 antibody, after cutting into slices to 60 DEG C of phosphate buffer affected tissues and checking discovery tissue slice redfree color product residual, repeat above-mentioned steps and carry out CD5, CD7, CD8, CD10, CD15, CD30, CD56, these several antigenic marks of CD20 dye, after completing, repeat above-mentioned steps and carry out the antigenic mark of CD3, then, carry out routine hematoxylin-eosin stains, resinene mounting, tissue slice is scanned under ordinary optical microscope.The scanned picture of the above-mentioned tissue slice of relative analysis, display positive expression is shown in red, first time carries out the intensity of CD3 labelling dyeing and the strength similarity of last CD3 labelling dyeing, analyze all pictorial informations, illustrate the expression of each cell these 10 kinds of antigens of CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD30, CD56, CD20 on this tissue slice.
Method two: by the flesh tissue specimen of lymph node puncture, conventional OCT embedding, freezing microtome section, picking up a tissue slice 4(thickness with band groove microscope slide 1 is 4 μm) and it is positioned over groove central authorities, coverslip 3 is placed in groove 2 and covers tissue slice 4.Phosphate buffer washes one time, monoclonal CD3 antibody working solution 100 microlitre is added into the gap place between coverslip and groove, hatch 60 minutes for 37 DEG C, phosphate buffer washes three times, every all over 5 minutes, add fluorescently-labeled second antibody, hatch 30 minutes for 37 DEG C, phosphate buffer washes three times, every all over 5 minutes, tissue slice is scanned under fluorescence microscope, 60 DEG C of phosphate buffer affected tissues are cut into slices 5 minutes, room temperature phosphate buffer washes three times, every all over 5 minutes, check under fluorescence microscope, if finding fluorescence residual, repeatable 60 DEG C of phosphate buffer affected tissues are cut into slices 5 minutes, check carries out next and plants antigenic mark dyeing after finding tissue slice unstressed configuration residual；Monoclonal CD4 antibody working solution 100 microlitre is added into the gap place between coverslip and groove, repeat the step after adding monoclonal CD3 antibody, after cutting into slices to 60 DEG C of phosphate buffer affected tissues and checking discovery tissue slice unstressed configuration residual, repeat above-mentioned steps and carry out the dyeing of these several antigenic marks of CD5, CD7, CD8, CD10, CD15, CD30, CD56, CD20, after completing, repeat the antigenic mark dyeing carrying out CD3, then, carry out routine hematoxylin-eosin stains, resinene mounting, scans tissue slice under ordinary optical microscope.The scanned picture of the above-mentioned tissue slice of relative analysis, display positive expression cell has fluorescent material to adhere to, first time carries out the intensity of CD3 labelling dyeing and the strength similarity of last CD3 labelling dyeing, analyze all pictorial informations, the expression of each cell these 10 kinds of antigens of CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD30, CD56, CD20 on this tissue slice can be illustrated.
Claims (5)
1. for same tissue slice being carried out repeatedly the apparatus and method of immunostaining, including tissue slice bogey and repeatedly staining, it is characterised in that:
Described tissue slice bogey, including band groove microscope slide and coverslip two parts, described it is made up of the flint glass of high transparent with groove microscope slide, there are a groove in central authorities, groove-bottom is smooth, according to the described coverslip size intending coupling, the level that is fabricated to by groove puts into coverslip, and described coverslip is commercially available conventional coverslip；
Described repeatedly staining, prepare before first carrying out the dyeing of routine, including the section of paraffin-embedded tissue, drift sheet, tissue slice is placed in the bottom portion of groove central authorities with groove microscope slide, a series of conventional steps (frozen section can dye after then cutting into slices) such as roasting sheet, dewaxing, aquation, antigen retrieval, coverslip is placed in above tissue slice, the dyeing that then tissue slice circulated successively, the repeatedly dyeing taken pictures, clean, dye again, take pictures again, clean again.
2. the apparatus and method for same tissue slice being carried out repeatedly immunostaining according to claim 1, it is characterised in that: described tissue slice bogey, in repeatedly staining implementation process, coverslip continues to cover tissue slice.
3. the apparatus and method for same tissue slice being carried out repeatedly immunostaining according to claim 1, it is characterized in that: described repeatedly staining, by the photo obtained after repeatedly dyeing is compared, thus obtaining the corresponding antigens express spectra of each cell on tissue slice.
4. the apparatus and method for same tissue slice being carried out repeatedly immunostaining according to claim 1, it is characterized in that: described repeatedly staining, adopt the phosphate buffer (about 60 DEG C) of heat to remove the first antibody and the second antibody that are attached to tissue slice.
5. the apparatus and method for same tissue slice being carried out repeatedly immunostaining according to claim 1, it is characterised in that: described repeatedly staining, with AEC (3-amino-9-ethylcarbazole；AEC), time as colour reagent, color product is washed away with dehydrated alcohol and dimethylbenzene.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106289923A (en) * | 2016-09-29 | 2017-01-04 | 南昌德漫多科技有限公司 | A kind of device for same tissue slice being carried out repeatedly immunostaining |
| CN106979878A (en) * | 2017-05-09 | 2017-07-25 | 南昌德漫多科技有限公司 | A kind of histopathology sample processing system |
| CN109612798A (en) * | 2018-12-25 | 2019-04-12 | 宁波察微生物科技有限公司 | A kind of glass slide dyeing, mounting, scanning integrated machine |
| CN111273007A (en) * | 2020-03-13 | 2020-06-12 | 内江师范学院 | Kit and detection method for rapidly detecting fish Edwardsiella ictaluri |
| CN111766125A (en) * | 2020-07-29 | 2020-10-13 | 广州金域医学检验中心有限公司 | Staining method using fluorescence quenching time difference, automatic staining apparatus, device, and medium |
| CN113390666A (en) * | 2021-06-17 | 2021-09-14 | 中国科学技术大学 | Method for detecting performance index of chemical substance in cell |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106289923A (en) * | 2016-09-29 | 2017-01-04 | 南昌德漫多科技有限公司 | A kind of device for same tissue slice being carried out repeatedly immunostaining |
| CN106979878A (en) * | 2017-05-09 | 2017-07-25 | 南昌德漫多科技有限公司 | A kind of histopathology sample processing system |
| CN106979878B (en) * | 2017-05-09 | 2023-12-15 | 贵阳德漫多医疗科技有限公司 | Histopathological specimen processing system |
| CN109612798A (en) * | 2018-12-25 | 2019-04-12 | 宁波察微生物科技有限公司 | A kind of glass slide dyeing, mounting, scanning integrated machine |
| CN111273007A (en) * | 2020-03-13 | 2020-06-12 | 内江师范学院 | Kit and detection method for rapidly detecting fish Edwardsiella ictaluri |
| CN111273007B (en) * | 2020-03-13 | 2023-08-01 | 内江师范学院 | Kit for rapidly detecting Edwardsiella ictaluri and detection method |
| CN111766125A (en) * | 2020-07-29 | 2020-10-13 | 广州金域医学检验中心有限公司 | Staining method using fluorescence quenching time difference, automatic staining apparatus, device, and medium |
| CN113390666A (en) * | 2021-06-17 | 2021-09-14 | 中国科学技术大学 | Method for detecting performance index of chemical substance in cell |
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Application publication date: 20160720 |