CN105067813A - Method for rapidly detecting T-synthase activity - Google Patents
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Abstract
The invention relates to a field of biomedicine, and concretely relates to an immunohistochemistry and immunofluorescence method for detecting galactosyltransferase, namely,a technology for detecting T-synthase activity. According to the invention, the immunohistochemistry or immunofluorescence detection method is used for rapidly and accurately determining a pathology characteristic of tissue cells with T-synthase inactivated, namely, Tn antigens and STn antigens being double positive. Under the condition, the Tn antigens and the STnantigenshave good and stable expression, and can be detected easily.
Description
Technical field
The present invention relates to biomedical sector, be specifically related to the method for one immunohistochemistry and immuno-fluorescence assay galactosyltransferase T-synthase (core1 β 3-galactosyltransferase, T-synthase) activity.
Background technology
T-synthase (T-synthase) is unique glycosyl transferase of synthesis core 1 structure, and its main function is added on GalNAc α 1-Ser/Thr (Tn antigen) sugar chain by galactose (Galactose).In people and other vertebrates; T-synthase inactivation will cause body cell can only synthesize Tn antigen and sialylated Tn (sialylTn; STn; Neu5Ac α 2; 6GalNAc α 1-O-Ser/Thr), but independent tumour glycoprotein antigen Tn or STn expresses, and do not mean that the forfeiture of T-synthase activity; during contrary Tn increasing expression sometimes, raise with T-synthase activity is abnormal.Whether normal the activity of T-synthase is occur with cancer and the disease such as Tn-syndrome closely related.
Tn antigen, chemical name GalNAc α 1-Ser/Th, also known as CD175; STn antigen, chemical name Neu5Ac α 2,6GalNAc α 1-O-Ser/Thr, also known as sialylTn, CD175s and CA72-4; Chinese CA72-4, also known as CA72-4, cancer antigen 72-4.
At present, more existing detection meanss in existing Protocols in Molecular Biology, but high cost, trivial operations and have certain pollution (as isotope), therefore apply limited.For this situation, foreign scholar proposes a kind of new detection method, the title of original text is AnovelfluorescentassayforT-synthaseactivity, document is published on " Glycobiology " volume21 (3) p352-362 of 2010, although the method avoids use isotope, but fresh histocyte can only be used for, and the detection of histopathologic slide can not be used for.
Summary of the invention
In order to solve the problem, the invention discloses the technology of qualitative detection T-synthase activity efficiently.
Technical scheme of the present invention is, with immunohistochemistry double-staining or with immunohistochemistry, detects histopathologic slide Tn and STn antigen simultaneously, when this histotomy Tn and STn expresses two positive, namely shows this cell T-synthase inactivation.
Wherein, immunohistochemistry double-staining operation steps is as follows,
Step one, dewaxing aquation: successively microslide is put into dimethylbenzene and graded ethanol.
Step 2, antigen retrieval: rinse 5 minutes in clear water after dewaxing, then add damping fluid and carry out heating 10-30 minute, come out in the site of antigen.
Step 3, site are closed: outwelled by damping fluid, add serum, some nonspecific sites are closed, and 37 ° of C hatch half an hour.Step 4, add two kinds of primary antibodies: the specific antibody namely dripping anti-Tn and STn antigen, 4 ° of C spend the night simultaneously.Step 5, add two and resist: microslide is taken out from refrigerator, puts into PBS and wash 3 times, each 5min, add after drying the PBS around tissue and resist (respectively anti-Tn antibody and anti-STn antibody) in conjunction with two of hydrogen peroxidase and alkaline phosphatase respectively.Then 37 ° of C incubator half an hour are placed in.
Step 6, add developer: taken out from incubator by slice, thin piece, put into PBS and wash 3 times, each 5min, after drying the PBS around tissue, add developer.Now, the Color Appearance System of alkaline phosphatase is BCIP/NBI; The Color Appearance System of peroxidase is AEC.
Step 7, to redye: the slice, thin piece clear water after colour developing is rinsed 5 minutes, and be soaked in haematine and dye, general animal tissue is half a minute.
Immunohistochemistry, specifically comprises the steps:
The preparation of step one, paraffin section: dewaxing aquation, antigen retrieval are with immunohistochemistry double-staining, and then need to dry for frozen section and cell climbing sheet, 4% paraformaldehyde fixes 10 ~ 15min.Then, PBS liquid washes 3 times, each 5 minutes.
Step 2, site are closed: the PBS using the normal second antibody source of species serum containing 5%, hatch section 60 minutes for 37 DEG C.
Step 3, first antibody, the i.e. antibody of Tn and STn antigen: in section, blot unnecessary confining liquid, to be correspondingly mixed incubated overnight with the unlabelled first antibody of two species diluted.When being used for direct immunization dyeing with fluorescently-labeled original antibodies, then can skip the step of the indirect immuning dyeing method by second antibody.3 × 5 minutes are washed in PBS liquid or TBS.
Step 4, second antibody, the i.e. antibody of anti-Tn with STn antibody: by a pair different fluorescein-labeled second antibody, in order to the original antibodies in conjunction with corresponding species, at room temperature hatch section 60 minutes, wash 3 × 5 minutes in PBS.
Step 5, to redye: carry out nuclear staining if necessary, as with DAPI, in PBS, wash section 3 × 5 minutes.
Step 6, selectivity: in order to stop fluorescence labeling to depart from from antibody, also in order to longer preservation is to protect dyeing, before aqueous medium uses, be recommended in the PBS liquid containing 4% formaldehyde and process 1-3 minute.Section is washed 2 times, each 2-3 minute in PBS.
Step 7, mounting are observed: section drips the anti-medium that fades, at the fluorescence microscopy Microscopic observation of different wave length.
Beneficial effect: the present invention is by the detection method of immunohistochemistry and immunofluorescence, determine the histiocytic pathological characteristics of T-synthase (T-synthase) inactivation fast and accurately, namely Tn and STn antigen is two positive, in such cases, Tn and STn antigen has high stability expression on tumor cell membrane, is easy to detect.The method, for the effect of research T-synthase, especially has important meaning with the relation of disease.
Embodiment
In order to better set forth innovation main points of the present invention, below with specific embodiment further instruction.
The method of the detection T-synthase inactivation of embodiment 1 the present embodiment is that concrete steps are as follows with immunohistochemistry double-staining:
Step one, dewaxing aquation: microslide is put into dimethylbenzene and graded ethanol;
Step 2, antigen retrieval: rinse 5 minutes in clear water after dewaxing, add 3%H
2o
2soak 10min, thus remove endogenic hydrogen peroxidase, then outwell H
2o
2, in clear water, wash twice, then add damping fluid and carry out heating 10-30 minute, come out in the site of antigen.
Step 3, site are closed: outwelled by damping fluid, add serum, some nonspecific sites are closed, 37 DEG C of half an hour.Step 4, add two kinds of primary antibodies: the specific antibody namely dripping anti-Tn and STn antigen, 4 DEG C are spent the night simultaneously.Step 5, add two and resist: microslide is taken out from refrigerator, puts into PBS and wash 3 times, each 5min, add after drying the PBS around tissue and resist (respectively anti-Tn antibody and anti-STn antibody) in conjunction with two of hydrogen peroxidase and enzyme respectively.Then 37 ° of C incubator half an hour are placed in.
Step 6, add developer: taken out from incubator by slice, thin piece, put into PBS and wash 3 times, each 5min, after drying the PBS around tissue, add developer.
Step 7, to redye: the slice, thin piece clear water after colour developing is rinsed 5 minutes, and be soaked in haematine and dye, animal tissue is generally half a minute.
Embodiment 2, the present embodiment be by immunohistochemistry to detect the activity of T-synthase, specifically comprise the steps:
The preparation of step one, paraffin section: dewaxing aquation, antigen retrieval, then need to dry for frozen section and cell climbing sheet, 4% paraformaldehyde fixes 10 ~ 15min.Then, PBS liquid washes 3 times, each 5 minutes.
Step 2, site are closed: hatch section 60 minutes (37 DEG C) with the PBS of the normal second antibody source of species serum containing 5%.
Step 3, first antibody, the i.e. antibody of Tn and STn antigen: in section, blot unnecessary confining liquid, correspondingly with two species of dilution, as mouse and rabbit, unlabelled first antibody is mixed incubated overnight, washes 3 × 5 minutes in PBS liquid or TBS.When being used for direct immunization dyeing with fluorescently-labeled original antibodies, then can skip the step of the indirect immuning dyeing method by second antibody.
Step 4, second antibody, the i.e. antibody of anti-Tn with STn antibody: by a pair different fluorescein-labeled second antibody, in order to the original antibodies in conjunction with corresponding species, at room temperature hatch section 60 minutes, wash 3 × 5 minutes in PBS.
Step 5, to redye: carry out nuclear staining if necessary, as with DAPI, in PBS, wash section 3 × 5 minutes.
Step 6, selectivity: in order to stop fluorescence labeling to depart from from antibody, also in order to longer preservation is to protect dyeing, before aqueous medium uses, be recommended in the PBS liquid containing 4% formaldehyde and process 1-3 minute.Section is washed 2 times, each 2-3 minute in PBS.
Step 7, mounting are observed: section drips the anti-medium that fades, at the fluorescence microscopy Microscopic observation of different wave length.
More than show and describe ultimate principle of the present invention and advantage.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and instructions just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (5)
1. one kind is detected the method for T-synthase activity fast, it is characterized in that, with immunohistochemistry double-staining or with immunohistochemistry, detect Tn and STn antigen in histopathologic slide simultaneously, when Tn and STn expresses two positive on the cell membrane of this histotomy, namely show this cell T-synthase inactivation.
2. the method for a kind of quick detection T-synthase activity according to claim 1, it is characterized in that, the concrete steps of immunohistochemistry double-staining are as follows:
Step one, dewaxing aquation: successively microslide is put into dimethylbenzene and graded ethanol;
Step 2, antigen retrieval: rinse 5 minutes in clear water after dewaxing, add 3%H
2o
2soak 10min, thus remove endogenic hydrogen peroxidase, then outwell H
2o
2, in clear water, wash twice, then add damping fluid and heat, come out in the site of antigen;
Step 3, site are closed: outwelled by damping fluid, add serum, some nonspecific sites are closed, and 37 ° of C hatch half an hour;
Step 4, add two kinds of original antibodies, be called for short primary antibodie: the specific antibody namely dripping anti-Tn and STn antigen, 4 ° of C spend the night simultaneously;
Step 5, add the antibody of anti-Tn and STn antibody simultaneously, be called for short two to resist: microslide is taken out from refrigerator, put into PBS and wash 3 times, each 5min, add respectively after drying the PBS around tissue connect from different substrates enzymes two resist, i.e. peroxidase-anti-Tn antibody and alkaline phosphatase-anti-STn antibody; Then 37 ° of C incubator half an hour are placed in;
Step 6, add developer: taken out from incubator by slice, thin piece, put into PBS and wash 3 times, each 5min, after drying the PBS around tissue, add developer;
Step 7 is redyed: after the slice, thin piece clear water after colour developing is rinsed a period of time, be soaked in haematine and dye, animal tissue is generally half a minute.
3. the method for a kind of quick detection T-synthase activity according to claim 2, its
Be characterised in that, described step 6 developer: the Color Appearance System of alkaline phosphatase is BCIP/NBI; The Color Appearance System of peroxidase is AEC.
4. the method for a kind of quick detection T-synthase activity according to claim 1, its
Be characterised in that, immunohistochemistry concrete steps are as follows:
The preparation of step one, paraffin section: dewaxing aquation, antigen retrieval are with immunohistochemistry double-staining, and then need to dry for frozen section and cell climbing sheet, 4% paraformaldehyde fixes 10 ~ 15min; Then, PBS liquid washes 3 times, each 5 minutes;
Step 2, site are closed: the PBS using the normal second antibody source of species serum containing 5%, hatch section 60 minutes for 37 DEG C;
Step 3, first antibody, the i.e. antibody of Tn and STn antigen: in section, blot unnecessary confining liquid, to be correspondingly mixed incubated overnight with the unlabelled first antibody of two species diluted; 3 times are washed, each 5 minutes in PBS liquid or TBS;
Step 4, second antibody, the i.e. antibody of anti-Tn with STn antibody: by a pair different fluorescein-labeled second antibody, in order to the original antibodies in conjunction with corresponding species, at room temperature hatch section 60 minutes, wash 3 × 5 minutes in PBS;
Step 5, to redye: carry out nuclear staining if necessary, as with DAPI, in PBS, wash section 3 × 5 minutes;
Step 6, selectivity: in order to stop fluorescence labeling to depart from from antibody, also in order to longer preservation is to protect dyeing, before aqueous medium uses, be recommended in the PBS liquid containing 4% formaldehyde and process 1-3 minute;
Section is washed 2 times, each 2-3 minute in PBS;
Step 7, mounting are observed: section drips the anti-medium that fades, at the fluorescence microscopy Microscopic observation of different wave length.
5. the method for a kind of quick detection T-synthase activity according to claim 4, in described step 3, two species are selected from rabbit and mouse.
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| CN107677827A (en) * | 2017-10-09 | 2018-02-09 | 亚能生物技术（深圳）有限公司 | A kind of double transfection reagent boxes of cervical carcinoma supplementary classification diagnosis and its application |
| CN108196057A (en) * | 2017-12-22 | 2018-06-22 | 青岛市农业科学研究院 | A kind of method for observing large biological molecule distribution and application |
| CN110161241A (en) * | 2019-04-30 | 2019-08-23 | 丁晓昆 | A kind of quickly detection biological tissue section COSMC gene mutation kit |
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| CN110161241A (en) * | 2019-04-30 | 2019-08-23 | 丁晓昆 | A kind of quickly detection biological tissue section COSMC gene mutation kit |
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