CN110501498A - A kind of p16 immunologic combined detection reagent kit - Google Patents
A kind of p16 immunologic combined detection reagent kit Download PDFInfo
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 32
- 230000001900 immune effect Effects 0.000 title claims abstract description 23
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 36
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 239000000243 solution Substances 0.000 claims abstract description 30
- 229960002685 biotin Drugs 0.000 claims abstract description 21
- 239000011616 biotin Substances 0.000 claims abstract description 21
- 239000007853 buffer solution Substances 0.000 claims abstract description 21
- 239000000427 antigen Substances 0.000 claims abstract description 20
- 102000036639 antigens Human genes 0.000 claims abstract description 20
- 108091007433 antigens Proteins 0.000 claims abstract description 20
- 239000012224 working solution Substances 0.000 claims abstract description 20
- 235000020958 biotin Nutrition 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 18
- 238000002372 labelling Methods 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 239000000872 buffer Substances 0.000 claims abstract description 15
- 238000003364 immunohistochemistry Methods 0.000 claims abstract description 14
- 238000011534 incubation Methods 0.000 claims abstract description 14
- 239000012188 paraffin wax Substances 0.000 claims abstract description 12
- 239000000843 powder Substances 0.000 claims abstract description 10
- 239000001509 sodium citrate Substances 0.000 claims abstract description 9
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 9
- 235000011330 Armoracia rusticana Nutrition 0.000 claims abstract description 7
- 240000003291 Armoracia rusticana Species 0.000 claims abstract description 7
- 108090001008 Avidin Proteins 0.000 claims abstract description 7
- 238000004043 dyeing Methods 0.000 claims abstract description 5
- 150000001412 amines Chemical class 0.000 claims abstract description 4
- 239000002981 blocking agent Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 229940088598 enzyme Drugs 0.000 claims description 13
- 239000012895 dilution Substances 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 12
- 241000283707 Capra Species 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 238000009835 boiling Methods 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 5
- 229940027941 immunoglobulin g Drugs 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 241000700198 Cavia Species 0.000 claims description 3
- 102000016943 Muramidase Human genes 0.000 claims description 3
- 108010014251 Muramidase Proteins 0.000 claims description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 3
- 102000003992 Peroxidases Human genes 0.000 claims description 3
- 210000004381 amniotic fluid Anatomy 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 229960000274 lysozyme Drugs 0.000 claims description 3
- 235000010335 lysozyme Nutrition 0.000 claims description 3
- 239000004325 lysozyme Substances 0.000 claims description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 239000003643 water by type Substances 0.000 claims description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 150000001299 aldehydes Chemical class 0.000 description 6
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
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- 238000004132 cross linking Methods 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 238000012744 immunostaining Methods 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
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- 229920002866 paraformaldehyde Polymers 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical group [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 150000002500 ions Chemical group 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007787 solid Substances 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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- G01N2001/302—Stain compositions
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
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- Food Science & Technology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to biotechnology technical field, specially a kind of p16 immunologic combined detection reagent kit, including primary antibody: freeze-dried powder p16 antibody；Without color reagent: endogenous peroxydase blocking agent 10-15ml；Reagent A: immunohistochemistry confining liquid；Reagent B: biotin labeling secondary antibody working solution；Reagent C: horseradish enzyme marks chain enzyme avidin working solution；Brown developing solution D liquid 1-2ml；White developing solution E liquid 20-25ml；0.1M PBS buffer solution；Sodium citrate antigen retrieval buffers；Diamino joins this amine DAB dyeing liquor.The present invention utilizes Streptavidin-biotin signal amplifying technique production immunohistochemistry detection reagent, with high sensitivity, high specificity, qualitative positioning are accurate, clear background, low power scape dye, directly it is added dropwise, it is easy to operate, incubation time is short, suitable for frozen section, paraffin section and the cell smear of fixation；It can be used for detecting cell, the specific antigen in tissue.
Description
Technical field
The present invention relates to biotechnology technical field, specially a kind of p16 immunologic combined detection reagent kit.
Background technique
Immunohistochemistry is to make the colour developing of labelled antibody by chemically reacting using principle of the antigen in conjunction with antibody specificity
Agent (fluorescein, enzyme, metal ion, isotope) develops the color to determine histocyte endoantigen (peptide and protein), carries out to it
It is positioning, qualitative and determine quantifier elimination.Immunohistochemistry has been widely used at present diagnoses with clinicopathologia, especially with being directed to
Property antibody carry out tumour auxiliary diagnosis and antidiastole, become clinicopathologia and diagnose indispensable important means.
P16 immunologic combined detection reagent kit sensitivity at present on the market is low, poor specificity, qualitative positioning inaccuracy, back
Scape is fuzzy, inconvenient for operation, and incubation time is too long.In consideration of it, we provide a kind of p16 immunologic combined detection reagent kit.
Summary of the invention
The purpose of the present invention is to provide a kind of p16 immunologic combined detection reagent kits, to solve to mention in above-mentioned background technique
Nowadays the sensitivity of p16 immunologic combined detection reagent kit is low out, and poor specificity, qualitative positioning inaccuracy, blurred background, operation are not
It is convenient, the too long problem of incubation time.
To achieve the above object, the invention provides the following technical scheme:
A kind of p16 immunologic combined detection reagent kit includes following reagent in the detection kit:
Primary antibody: freeze-dried powder p16 antibody；
Without color reagent: endogenous peroxydase blocking agent 10-15ml；
Reagent A: immunohistochemistry confining liquid；
Reagent B: biotin labeling secondary antibody working solution；
Reagent C: horseradish enzyme marks chain enzyme avidin working solution；
Brown developing solution D liquid 1-2ml；
White developing solution E liquid 20-25ml；
0.1M PBS buffer solution；
Sodium citrate antigen retrieval buffers；
Diamino joins this amine DAB dyeing liquor.
Preferably, the experimental procedure of the detection kit is specific as follows:
S1: primary antibody dilution and DAB developing solution are prepared；
S2: it by paraffin section, and dewaxes to water；
S3: distilled water flushing, 0.1M PBS buffer solution impregnate 6-10min；
S4: antigen retrieval is carried out；
S5: it is incubated for 15-20min using colourless double amniotic fluid deionized waters of 3%-5%, it is living to eliminate endogenous peroxidase
Property；
S6: being added dropwise blue agents A immunohistochemistry confining liquid, is incubated at room temperature 15-20min；
S7: being added dropwise the primary antibody dilution for preparing in S1,35-37 DEG C incubation 2-3 hours；
S8: being rinsed using 0.1M PBS buffer solution, is rinsed once within each minute, is flushed three times within three minutes, is added dropwise later yellow
Color reagent B biotin labeling secondary antibody working solution, 30-37 DEG C of incubation 15-20min；
S9: being rinsed using 0.1M PBS buffer solution, is rinsed once within each minute, is flushed three times within three minutes, orange is added dropwise later
Color reagent C horseradish enzyme marks chain enzyme avidin working solution, 30-37 DEG C of incubation 15-20min；
S10: being developed the color by DAB developing solution, and is sufficiently rinsed by tap water；
S11: being redyed, be dehydrated, is transparent, selects mountant mounting appropriate later.
Preferably, the primary antibody dilution is diluted using following two method:
(1) freeze-dried powder p16 antibody 0.1M PBS buffer solution is diluted, the antibody after dilution is dispensed into 5-10 branch,
20ulx5 branch or 10ulx10 branch, are put into -20 DEG C of preservations；
(2) by freeze-dried powder p16 antibody with 0.1M PBS buffer solution dilute 50ul at stoste, then plus 50% glycerol, be put into-
20 DEG C of preservations.
Preferably, the restorative procedure of the sodium citrate antigen retrieval buffers includes boiling water bath reparation, Microwave method or height
Pressure is repaired.
Preferably, the preparation experiment of the DAB developing solution specifically comprises the following steps: the white developing solution E liquid of 1ml
As DAB substrate solution, the brown developing solution D liquid of 1 drop 50ul is added later as DAB concentrate, is uniformly mixed, it is aobvious that DAB is made
Color liquid.
Preferably, the immunohistochemistry confining liquid includes closing Normal Goat Serum working solution.
Preferably, the biotin labeling secondary antibody working solution uses biotin labeling goat anti-rabbit immunoglobulin G, biology
Element label goat anti-mouse immunoglobulin G or biotin labeling goat-anti rabbit, rat, mouse and immunized guinea pigs Lysozyme.
Preferably, the pH value of the PBS buffer solution is pH7.2-7.4.
Preferably, sterile distilled water can also be used in the PBS buffer solution.
Compared with prior art, the beneficial effects of the present invention are:
1, this p16 immunologic combined detection reagent kit is immune using Streptavidin-biotin signal amplifying technique production
Groupization detection reagent has high sensitivity, and high specificity, qualitative positioning are accurate, clear background, low power scape dye, and is directly added dropwise,
Easy to operate, incubation time is short, suitable for frozen section, paraffin section and the cell smear of fixation；It can be used for detecting cell, group
Knit interior specific antigen.
2, this p16 immunologic combined detection reagent kit is capable of providing metastable ion ring by the PBS buffer solution being arranged
Border and pH buffer capacity are the buffer salt solutions being commonly used in biology, and for molecular cloning and cell culture etc., pH is
7.2-7.4, isotonic with blood of human body, main component is potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride.
3, this p16 immunologic combined detection reagent kit can be used for paraffin and cut by the sodium citrate antigen retrieval buffers being arranged
Antigen retrieval of the samples such as piece, frozen section using paraformaldehyde, formaldehyde or other aldehydes reagents after fixed, can effectively remove
Crosslinking between albumen caused by aldehydes fixating reagent, the sufficiently epitope in the samples such as exposure paraffin section, thus significantly
Improve immunostaining effect.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
A kind of p16 immunologic combined detection reagent kit includes following reagent in detection kit:
Primary antibody: freeze-dried powder p16 antibody；
Without color reagent: endogenous peroxydase blocking agent 10-15ml；
Reagent A: immunohistochemistry confining liquid；
Reagent B: biotin labeling secondary antibody working solution；
Reagent C: horseradish enzyme marks chain enzyme avidin working solution；
Brown developing solution D liquid 1-2ml；
White developing solution E liquid 20-25ml；
0.1M PBS buffer solution；
Sodium citrate antigen retrieval buffers；
Diamino joins this amine DAB dyeing liquor.
PBS (phosphate buffered solution) i.e. phosphate buffer, is capable of providing metastable ion
Environment and pH buffer capacity are the buffer salt solutions being commonly used in biology, and for molecular cloning and cell culture etc., pH is
7.2-7.4, isotonic with blood of human body, main component is potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride etc..
The detection kit experimental procedure of the present embodiment is specific as follows:
S1: primary antibody dilution and DAB developing solution are prepared；
S2: it by paraffin section, and dewaxes to water；
S3: distilled water flushing, 0.1M PBS buffer solution impregnate 6-10min；
S4: antigen retrieval is carried out；
S5: it is incubated for 15-20min using colourless double amniotic fluid deionized waters of 3%-5%, it is living to eliminate endogenous peroxidase
Property；
S6: being added dropwise blue agents A immunohistochemistry confining liquid, is incubated at room temperature 15-20min；
S7: being added dropwise the primary antibody dilution for preparing in S1,35-37 DEG C incubation 2-3 hours；
S8: being rinsed using 0.1M PBS buffer solution, is rinsed once within each minute, is flushed three times within three minutes, is added dropwise later yellow
Color reagent B biotin labeling secondary antibody working solution, 30-37 DEG C of incubation 15-20min；
S9: being rinsed using 0.1M PBS buffer solution, is rinsed once within each minute, is flushed three times within three minutes, orange is added dropwise later
Color reagent C horseradish enzyme marks chain enzyme avidin working solution, 30-37 DEG C of incubation 15-20min；
S10: being developed the color by DAB developing solution, and is sufficiently rinsed by tap water；
S11: being redyed, be dehydrated, is transparent, selects mountant mounting appropriate later.
Immunohistochemistry confining liquid includes closing Normal Goat Serum working solution.
Biotin labeling secondary antibody working solution is anti-using biotin labeling goat anti-rabbit immunoglobulin G, biotin labeling goat
Mouse immune globulin G or biotin labeling goat-anti rabbit, rat, mouse and immunized guinea pigs Lysozyme.
Further, the pH value of PBS buffer solution is pH7.2-7.4, so that pH value is moderate.
In addition, sterile distilled water can also be used in PBS buffer solution, cost can be reduced.
The p16 immunologic combined detection reagent kit of the present embodiment is produced using Streptavidin-biotin signal amplifying technique
Immunohistochemistry detection reagent, there is high sensitivity, high specificity, qualitative positioning be accurate, the dyeing of clear background, low power scape, directly
Dropwise addition is connect, easy to operate, incubation time is short, suitable for frozen section, paraffin section and the cell smear of fixation；It can be used for detecting
Specific antigen in cell, tissue；The PBS buffer solution of setting is capable of providing metastable ionic environment and pH buffer capacity
Power is the buffer salt solution being commonly used in biology, for molecular cloning and cell culture etc., pH 7.2-7.4, with people
Body blood is isotonic, and main component is potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride；Sodium citrate antigen retrieval buffers can be used for
Antigen retrieval of the samples such as paraffin section, frozen section using paraformaldehyde, formaldehyde or other aldehydes reagents after fixed, Ke Yiyou
Crosslinking between albumen caused by effect removal aldehydes fixating reagent, the sufficiently epitope in the samples such as exposure paraffin section, from
And substantially improve immunostaining effect.
Embodiment 2
As second of embodiment of the invention, primary antibody dilution is diluted using following two method:
(1) freeze-dried powder p16 antibody 0.1M PBS buffer solution is diluted, the antibody after dilution is dispensed into 5-10 branch,
20ulx5 branch or 10ulx10 branch, are put into -20 DEG C of preservations；
(2) by freeze-dried powder p16 antibody with 0.1M PBS buffer solution dilute 50ul at stoste, then plus 50% glycerol, be put into-
20 DEG C of preservations.
Specifically, DAB developing solution preparation experiment specifically comprise the following steps: using the white developing solution E liquid of 1ml as
DAB substrate solution is added the brown developing solution D liquid of 1 drop 50ul as DAB concentrate later, is uniformly mixed, DAB developing solution is made,
The necessary matching while using of this solution, is kept in dark place after preparing, uses in 6 hours, remaining liquid should discard, sample developing time one
As be room temperature 5-20 minute, observation colour developing situation, should be dehydrated in time sealing for sample after colour developing is abundant under the microscope.
Embodiment 3
The restorative procedure of sodium citrate antigen retrieval buffers includes boiling water bath reparation, Microwave method or Pressure method, citric acid
It is solid using paraformaldehyde, formaldehyde or other aldehydes reagents that sodium antigen retrieval buffers can be used for the samples such as paraffin section, frozen section
Antigen retrieval after fixed, can effectively remove the crosslinking between albumen caused by aldehydes fixating reagent, sufficiently expose paraffin section
Epitope in equal samples, to substantially improve immunostaining effect.
Specifically, boiling water bath reparation is placed in boiling water bath environment for the beaker for repairing liquid and slide is filled, external boiling is kept
State 15min, cooled to room temperature.
Microwave method will fill the beaker for repairing liquid and slide and be placed in micro-wave oven, Gao Huo 5min, truce 3min, moderate heat
5min, cooled to room temperature.
Pressure method will fill the beaker for repairing liquid and slide and be placed in pressure cooker, 2-5min is repaired after upper vapour, then will
Pressure cooker is placed in cold water, is decompressed to normal rear taking-up slide.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
For personnel it should be appreciated that the present invention is not limited to the above embodiments, described in the above embodiment and specification is only the present invention
Preference, be not intended to limit the invention, without departing from the spirit and scope of the present invention, the present invention also has various
Changes and improvements, these changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by institute
Attached claims and its equivalent thereof.
Claims (9)
1. a kind of p16 immunologic combined detection reagent kit, it is characterised in that: include following reagent in the detection kit:
Primary antibody: freeze-dried powder p16 antibody；
Without color reagent: endogenous peroxydase blocking agent 10-15ml；
Reagent A: immunohistochemistry confining liquid；
Reagent B: biotin labeling secondary antibody working solution；
Reagent C: horseradish enzyme marks chain enzyme avidin working solution；
Brown developing solution D liquid 1-2ml；
White developing solution E liquid 20-25ml；
0.1MPBS buffer；
Sodium citrate antigen retrieval buffers；
Diamino joins this amine DAB dyeing liquor.
2. p16 immunologic combined detection reagent kit according to claim 1, it is characterised in that: the reality of the detection kit
It is specific as follows to test step:
S1: primary antibody dilution and DAB developing solution are prepared；
S2: it by paraffin section, and dewaxes to water；
S3: distilled water flushing, 0.1MPBS buffer impregnate 6-10min；
S4: antigen retrieval is carried out；
S5: it is incubated for 15-20min using colourless double amniotic fluid deionized waters of 3%-5%, eliminates endogenous peroxidase activity；
S6: being added dropwise blue agents A immunohistochemistry confining liquid, is incubated at room temperature 15-20min；
S7: being added dropwise the primary antibody dilution for preparing in S1,35-37 DEG C incubation 2-3 hours；
S8: being rinsed using 0.1MPBS buffer, is rinsed once within each minute, is flushed three times within three minutes, yellow agents are added dropwise later
B biotin labeling secondary antibody working solution, 30-37 DEG C of incubation 15-20min；
S9: being rinsed using 0.1MPBS buffer, is rinsed once within each minute, is flushed three times within three minutes, orange reagent is added dropwise later
C horseradish enzyme marks chain enzyme avidin working solution, 30-37 DEG C of incubation 15-20min；
S10: being developed the color by DAB developing solution, and is sufficiently rinsed by tap water；
S11: being redyed, be dehydrated, is transparent, selects mountant mounting appropriate later.
3. p16 immunologic combined detection reagent kit according to claim 2, it is characterised in that: primary antibody dilution use with
Lower two methods are diluted:
(1) freeze-dried powder p16 antibody 0.1MPBS buffer is diluted, the antibody after dilution is dispensed into 5-10 branch, 20ulx5 branch
Or 10ulx10 branch, it is put into -20 DEG C of preservations；
(2) by freeze-dried powder p16 antibody with 0.1MPBS buffer dilute 50ul at stoste, then plus 50% glycerol, be put into -20 DEG C of guarantors
It deposits.
4. p16 immunologic combined detection reagent kit according to claim 2, it is characterised in that: the sodium citrate antigen is repaired
The restorative procedure of multiple liquid includes boiling water bath reparation, Microwave method or Pressure method.
5. p16 immunologic combined detection reagent kit according to claim 1, it is characterised in that: the preparation of the DAB developing solution
Experiment specifically comprises the following steps: using the white developing solution E liquid of 1ml as DAB substrate solution, and the brown of 1 drop 50ul is added later
Developing solution D liquid is uniformly mixed as DAB concentrate, and DAB developing solution is made.
6. p16 immunologic combined detection reagent kit according to claim 1, it is characterised in that: the immunohistochemistry confining liquid
Including closing Normal Goat Serum working solution.
7. p16 immunologic combined detection reagent kit according to claim 1, it is characterised in that: the biotin labeling secondary antibody
Working solution uses biotin labeling goat anti-rabbit immunoglobulin G, biotin labeling goat anti-mouse immunoglobulin G or biotin
Mark goat-anti rabbit, rat, mouse and immunized guinea pigs Lysozyme.
8. p16 immunologic combined detection reagent kit according to claim 1, it is characterised in that: the soda acid of the PBS buffer solution
Degree is pH7.2-7.4.
9. p16 immunologic combined detection reagent kit according to claim 1, it is characterised in that: the PBS buffer solution can also adopt
Use sterile distilled water.
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| CN112083164A (en) * | 2020-08-27 | 2020-12-15 | 檀碧波 | Kit for detecting Vav3 protein and application thereof |
| CN112180090A (en) * | 2020-10-09 | 2021-01-05 | 深圳市森盈生物科技有限公司 | Kit of monoclonal antibody specifically bound by immune cell p16 |
| CN112485426A (en) * | 2020-12-01 | 2021-03-12 | 山东省药物研究院 | Circulating tumor cell IHC antigen repairing method based on ISET principle |
| CN112816685A (en) * | 2020-12-30 | 2021-05-18 | 苏州堪赛尔医学检验有限公司 | Immunohistochemical immunoenzyme labeling method and kit for immunoenzyme labeling method |
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| CN112180090A (en) * | 2020-10-09 | 2021-01-05 | 深圳市森盈生物科技有限公司 | Kit of monoclonal antibody specifically bound by immune cell p16 |
| CN112485426A (en) * | 2020-12-01 | 2021-03-12 | 山东省药物研究院 | Circulating tumor cell IHC antigen repairing method based on ISET principle |
| CN112816685A (en) * | 2020-12-30 | 2021-05-18 | 苏州堪赛尔医学检验有限公司 | Immunohistochemical immunoenzyme labeling method and kit for immunoenzyme labeling method |
| CN113075194A (en) * | 2021-03-23 | 2021-07-06 | 武汉康义盛生物科技有限公司 | Novel Raman spectrum detection kit and use method thereof |
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