CN118091127A - Mammary gland cell immunochemistry staining kit and detection method - Google Patents
Mammary gland cell immunochemistry staining kit and detection method Download PDFInfo
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- 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
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Abstract
The invention discloses a mammary gland cell immunochemistry staining kit and a detection method, wherein the detection method comprises the following steps: step 1, preparing a staining solution; stain 1: PBS liquid, wherein the first antibody is rabbit anti-human estrogen receptor, rabbit anti-human progestogen receptor, cell nucleus coloring, rabbit anti-human CerbB-2 monoclonal antibody, cell membrane coloring, and the second antibody is quick immunity refinement Elivision kit and DAB kit; stain 2: nitrilotriacetic acid, gluconic acid, sorbitol, amino alcohol, paraformaldehyde, KC1 and buffer; step 2, tissue section: taking a breast specimen of a subject, fixing the breast specimen by neutral formaldehyde with the concentration of 10%, taking pathological tissues, and dehydrating the pathological tissues by alcohol gradient. The invention reduces the false positive influence of interfering substances in serum, improves the detection accuracy and specificity, improves the detection sensitivity, has high detection accuracy and low false negative rate, and has simple and convenient dyeing method operation, clear and visible dyeing result and easy interpretation.
Description
Technical Field
The invention relates to the technical field of kits, in particular to a mammary gland cell immunochemical staining kit and a detection method.
Background
Breast cancer is a common malignancy, occurring mainly in female breast tissue, but rarely occurs in men, and the following are some basic information for breast cancer: the reason is that: the specific cause of breast cancer is not completely understood, but may be related to various factors such as genetics, lifestyle and environmental factors. Certain genetic mutations and familial genetic predisposition can increase the risk of breast cancer. Other possible risk factors include age, hormone levels, obesity, poor diet, history of breast disease, long-term use of hormone replacement therapy, etc., symptoms: early breast cancer may have no obvious symptoms, but as the condition progresses, common symptoms may include breast lumps, breast skin changes (e.g., disharmony, skin thickening, skin redness, etc.), breast pain or discomfort, nipple discharge, breast swelling, lymph node swelling, etc., diagnosis: diagnosis of breast cancer typically includes mammography, breast ultrasound, mammography (molybdenum target or thallium mammography), and breast tissue biopsies, etc. These examinations help to determine the nature and stage of the lesions and guide the selection of subsequent treatment regimens, treatment: the treatment scheme of the breast cancer is various, including surgical excision, radiotherapy, chemotherapy, endocrine treatment, targeted treatment and the like. The specific treatment scheme is determined according to the individual condition, tumor characteristics and stage of the patient, and aims to achieve the aims of radical treatment, survival prolongation and life quality improvement, and prevention and early screening: preventive measures for breast cancer include maintaining a healthy lifestyle, periodic breast self-examination, periodic physical examination, participation in breast cancer screening programs, and the like. For high risk populations or populations with a significant familial genetic predisposition, more frequent screening and early detection may be required, it is important to discover and diagnose breast cancer early to begin appropriate treatment early and to work up a personalized treatment plan in close cooperation with the physician. If breast cancer is suspected, please consult with doctors in time to obtain professional advice and guidance, and the mammary gland cell immunity refinement staining is a common experimental technique for detecting and positioning the expression and distribution of specific proteins in mammary gland tissues, which can provide information about the types of mammary gland cells, proliferation activity, differentiation degree, protein expression level and the like;
The existing dyeing agent for the mammary gland cell immunochemistry staining method is easy to combine with non-target phosphorylated proteins, has more non-specific combination and background interference, is easy to generate false positive for interfering substances in serum, has low detection sensitivity and troublesome staining operation, and therefore, the invention provides a mammary gland cell immunochemistry staining kit and a detection method for solving the problems.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a mammary gland cell immunochemical staining kit and a detection method.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a mammary gland cell immunochemistry staining kit and a detection method, wherein the detection method comprises the following steps:
step 1, preparing a staining solution;
Stain 1: PBS liquid, wherein the first antibody is rabbit anti-human estrogen receptor, rabbit anti-human progestogen receptor, cell nucleus coloring, rabbit anti-human CerbB-2 monoclonal antibody, cell membrane coloring, and the second antibody is quick immunity refinement E1ivision kit and DAB kit;
Stain 2: nitrilotriacetic acid, gluconic acid, sorbitol, amino alcohol, paraformaldehyde, KC1 and buffer;
Step 2, tissue section: taking a breast specimen of a subject, fixing the breast specimen by neutral formaldehyde with the concentration of 10%, taking pathological tissues, dehydrating by alcohol gradient, soaking and embedding xylene in paraffin to prepare paraffin blocks, slicing the paraffin blocks, wherein the thickness of the paraffin blocks is between 2 and 3 mu m, spreading the slices to ensure that the slices are spread sufficiently, taking out the slices by adopting glass slides treated by APES anti-drop slices, and baking the slices in an incubator at the temperature of 65 ℃ for 2 to 4 hours;
Step 3, immune refinement staining: after baking the sheet is completed, fixing the tissue sample in a glass slide or a centrifuge tube to maintain the morphological structure and protect the target protein, performing infiltration treatment on the fixed tissue sample, using an infiltration agent, enabling an antibody to enter cells by Triton X-100, adding a pair of antibody solutions specifically binding to the target protein to the tissue sample, wherein one antibody can bind to a specific epitope of the protein, the other antibody is used for detecting the binding, washing the sample with a buffer solution to remove unbound antibodies and other non-specifically bound substances, amplifying the signal bound to the specific antibody by using a fluorescein or an enzyme substrate, observing the fluorescein usually by a fluorescence microscope, and allowing the enzyme substrate to form a color reaction under a microscope, observing the tissue sample by using a fluorescence microscope or a common optical microscope, recording the expression condition of the target protein for 15-20 minutes, and finally washing 3 times by using PBS (phosphate-buffered saline), performing DAB color development, counterstaining, and sealing.
The beneficial effects of the invention are as follows:
1. the immune refinement staining result shows that the pathological tissue structure is complete, no obvious flaking condition occurs, the negative and positive have clear contrast, the positive substance is positioned very accurately, the background is clean and very clear, the nonspecific staining is avoided, the result is stable and reliable, the sensitivity is high, and the specificity is also stronger;
2. The cell staining solution can rapidly repair cell antigens to fully expose antigenic determinants, and simultaneously improves cell membrane permeability, facilitates antibody molecules to enter cells to bind to the antigens, avoids binding with non-target phosphorylated proteins except Ki67 antibodies, enhances signal to noise ratio, and reduces non-specific binding and background interference; the false positive influence of interfering substances in serum is reduced, and the detection accuracy and specificity are improved; the stability of the Ki67 antibody is stabilized, the detection accuracy of the detection sensitivity is improved, the false negative rate is low, the dyeing method is simple and convenient to operate, and the dyeing result is clear and visible and easy to interpret.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments.
Example 1
A mammary gland cell immunochemistry staining kit and a detection method, the detection method comprises the following steps:
step 1, preparing a staining solution;
dyeing agent: PBS liquid, wherein the first antibody is rabbit anti-human estrogen receptor, rabbit anti-human progestogen receptor, cell nucleus coloring, rabbit anti-human CerbB-2 monoclonal antibody, cell membrane coloring, and the second antibody is quick immunity refinement Elivision kit and DAB kit;
Step 2, tissue section: taking a breast specimen of a subject, fixing the breast specimen by neutral formaldehyde with the concentration of 10%, taking pathological tissues, dehydrating by alcohol gradient, soaking and embedding xylene in paraffin to prepare paraffin blocks, slicing the paraffin blocks, wherein the thickness of the paraffin blocks is between 2 and 3 mu m, spreading the slices to ensure that the slices are spread sufficiently, taking out the slices by adopting glass slides treated by APES anti-drop slices, and baking the slices in an incubator at the temperature of 65 ℃ for 2 to 4 hours;
Step 3, immune refinement staining: after baking the sheet, fixing the tissue sample in a glass slide or a centrifuge tube by using a formaldehyde fixing agent to maintain the morphological structure of the tissue sample and protect target proteins, performing infiltration treatment on the fixed tissue sample, using the penetrating agent, enabling an antibody to enter cells by using a Triton X-100, adding a pair of antibody solutions specifically combined with the target proteins into the tissue sample, wherein one antibody can be combined with a specific epitope of the proteins, the other antibody is used for detecting the combination, washing the sample by using a buffer solution to remove unbound antibodies and other substances which are not specifically combined, amplifying signals combined with the specific antibodies by using fluorescein or an enzyme substrate, observing the fluorescein usually by using a fluorescence microscope, allowing the enzyme substrate to form a color reaction under a microscope, observing the tissue sample by using the fluorescence microscope or a common optical microscope, recording the expression condition of the target proteins for 15-20 minutes, and finally washing the sample by using PBS liquid for 3 times, performing DAB color development, counterstaining and sealing;
pET-23a (+) expression vector
Original sequence:
ATGGAGCCGGCGGCGGGGAGCAGCATGGAGCCTTCGGCTGACTGGCTGGCCACGGCCGCGGCCCGGGGTCGGGTAGAGGAGGTGCGGGCGCTGCTGGAGGCGGGGGCGCTGCCCAACGCACCGAATAGTTACGGTCGGAGGCCGATCCAGGTCATGATGATGGGCAGCGCCCGAGTGGCGGAGCTGCTGCTGCTCCACGGCGCGGAGCCCAACTGCGCCGACCCCGCCACTCTCACCCGACCCGTGCACGACGCTGCCCGGGAGGGCTTCCTGGACACGCTGGTGGTGCTGCACCGGGCCGGGGCGCGGCTGGACGTGCGCGATGCCTGGGGCCGTCTGCCCGTGGACCTGGCTGAGGAGCTGGGCCATCGCGATGTCGCACGGTACCTGCGCGCGGCTGCGGGGGGCACCAGAGGCAGTAACCATGCCCGCATAGATGCCGCGGAAGGTCCCTCAGACATCCCCGAT;
optimized gene sequence:
atgGCGAGCatgACCGGCGGCCAGCAGatgGGCCGTGGCAGCGAATTCatgGAACCGGCGGCGGGCAGCAGCatgGAACCGAGCGCGGATTGGCTGGCGACCGCGGCGGCGCGTGGCCGTGTGGAAGAAGTGCGTGCGCTGCTGGAAGCGGGCGCGCTGCCGAACGCGCCGAACAGCTATGGCCGTCGTCCGATTCAGGTGatgatgatgGGCAGCGCGCGTGTGGCGGAACTGCTGCTGCTGCATGGCGCGGAACCGAACTGCGCGGATCCGGCGACCCTGACCCGTCCGGTGCATGATGCGGCGCGTGAAGGCTTTCTGGATACCCTGGTGGTGCTGCATCGTGCGGGCGCGCGTCTGGATGTGCGTGATGCGTGGGGCCGTCTGCCGGTGGATCTGGCGGAAGAACTGGGCCATCGTGATGTGGCGCGTTATCTGCGTGCGGCGGCGGGCGGCACCCGTGGCAGCAACCATGCGCGTATTGATGCGGCGGAAGGCCCGAGCGATATTCCGGATAAGCTTGCGGCGGCGCTGGAACATCATCATCATCATCAT;
Amino acid sequence after optimization:
MASMTGGQQMGRGSEFMEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPDKLAAALEHHHHHH*;
The occurrence of yellow-stained particles on the cytoplasm or envelope of cancer cells is regarded as positive, wherein the particles appear pale yellow, and the uniform staining result is regarded as weak positive+; the particles appear yellow, and the distribution is less uniform, so that the staining result appears moderately positive++; the particles appear dark yellow or brown, and the blocky color is regarded as a strong positive++ as a staining result;
The mammary tissue section has no shrinkage or chapping phenomenon, the tissue structure cell morphology is clearly observed, breast cancer cells and inflammatory cells can be clearly distinguished, after HE staining, the cell nuclei are clearly blue, cytoplasm eosin staining is different in shade and pink, the cell nuclei are in blue with different degrees, the contrast is clear, the immune refining staining result shows that the pathological tissue structure is complete, no obvious flaking condition occurs, clear contrast exists between negative and positive, positive substances are quite accurate to position, the background is clean and quite clear, and no nonspecific staining exists. The result is stable and reliable, has high sensitivity and strong specificity.
Example two
A mammary gland cell immunochemistry staining kit and a detection method, wherein the detection method comprises the following steps:
step 1, preparing a staining solution;
dyeing agent: nitrilotriacetic acid, gluconic acid, sorbitol, amino alcohol, paraformaldehyde, KCl and buffers;
Step 2, tissue section: taking a breast specimen of a subject, fixing the breast specimen by neutral formaldehyde with the concentration of 10%, taking pathological tissues, dehydrating by alcohol gradient, soaking and embedding xylene in paraffin to prepare paraffin blocks, slicing the paraffin blocks, wherein the thickness of the paraffin blocks is between 2 and 3 mu m, spreading the slices to ensure that the slices are spread sufficiently, taking out the slices by adopting glass slides treated by APES anti-drop slices, and baking the slices in an incubator at the temperature of 65 ℃ for 2 to 4 hours;
Step 3, immune refinement staining: after baking the sheet is completed, fixing the tissue sample in a glass slide or a centrifuge tube with formaldehyde fixing agent to maintain the morphological structure and protect target proteins, performing infiltration treatment on the fixed tissue sample, using the penetrating agent, enabling an antibody to enter cells, adding a pair of antibody solutions specifically binding to the target proteins to the tissue sample, wherein one antibody can bind to a specific epitope of the proteins, the other antibody is used for detecting the binding, washing the sample with a buffer solution to remove unbound antibodies and other non-specifically bound substances, amplifying signals bound to the specific antibodies by using luciferin or an enzyme substrate, observing the luciferin usually by a fluorescence microscope, allowing the enzyme substrate to form a color reaction under a microscope, observing the tissue sample by using a fluorescence microscope or a common optical microscope, and recording the expression condition of the target proteins;
pET-22b (+) expression vector
Original sequence:
ATGGAGCCGGCGGCGGGGAGCAGCATGGAGCCTTCGGCTGACTGGCTGGCCACGGCCGCGGCCCGGGGTCGGGTAGAGGAGGTGCGGGCGCTGCTGGAGGCGGGGGCGCTGCCCAACGCACCGAATAGTTACGGTCGGAGGCCGATCCAGGTCATGATGATGGGCAGCGCCCGAGTGGCGGAGCTGCTGCTGCTCCACGGCGCGGAGCCCAACTGCGCCGACCCCGCCACTCTCACCCGACCCGTGCACGACGCTGCCCGGGAGGGCTTCCTGGACACGCTGGTGGTGCTGCACCGGGCCGGGGCGCGGCTGGACGTGCGCGATGCCTGGGGCCGTCTGCCCGTGGACCTGGCTGAGGAGCTGGGCCATCGCGATGTCGCACGGTACCTGCGCGCGGCTGCGGGGGGCACCAGAGGCAGTAACCATGCCCGCATAGATGCCGCGGAAGGTCCCTCAGACATCCCCGAT;
optimized gene sequence:
ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGAACCGGCGGCGGGCAGCAGCATGGAACCGAGCGCGGATTGGCTGGCGACCGCGGCGGCGCGCGGCCGCGTGGAAGAAGTGCGCGCGCTGCTGGAAGCGGGCGCGCTGCCGAACGCGCCGAACAGCTATGGCCGCCGCCCGATTCAGGTGATGATGATGGGCAGCGCGCGCGTGGCGGAACTGCTGCTGCTGCATGGCGCGGAACCGAACTGCGCGGATCCGGCGACCCTGACCCGCCCGGTGCATGATGCGGCGCGCGAAGGCTTTCTGGATACCCTGGTGGTGCTGCATCGCGCGGGCGCGCGCCTGGATGTGCGCGATGCGTGGGGCCGCCTGCCGGTGGATCTGGCGGAAGAACTGGGCCATCGCGATGTGGCGCGCTATCTGCGCGCGGCGGCGGGCGGCACCCGCGGCAGCAACCATGCGCGCATTGATGCGGCGGAAGGCCCGAGCGATATTCCGGATAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCAC;
Amino acid sequence after optimization:
MKYLLPTAAAGLLLLAAQPAMAMEPAAGSSMEPSADWLATAAARGRVEEVRALLEAGALPNAPNSYGRRPIQVMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRAGARLDVRDAWGRLPVDLAEELGHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPDKLAAALEHHHHHH*;
The cell staining solution can rapidly repair cell antigens to fully expose antigenic determinants, and simultaneously improves cell membrane permeability, facilitates antibody molecules to enter cells to bind to the antigens, avoids binding with non-target phosphorylated proteins except Ki67 antibodies, enhances signal to noise ratio, and reduces non-specific binding and background interference; the false positive influence of interfering substances in serum is reduced, and the detection accuracy and specificity are improved; the stability of the Ki67 antibody is stabilized, the detection accuracy of the detection sensitivity is improved, the false negative rate is low, the dyeing method is simple and convenient to operate, and the dyeing result is clear and visible and easy to interpret.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (1)
1. The mammary gland cell immunochemistry staining kit and the detection method are characterized in that the detection method comprises the following steps:
step 1, preparing a staining solution;
Stain 1: PBS liquid, wherein the first antibody is rabbit anti-human estrogen receptor, rabbit anti-human progestogen receptor, cell nucleus coloring, rabbit anti-human CerbB-2 monoclonal antibody, cell membrane coloring, and the second antibody is quick immunity refinement Elivision kit and DAB kit;
Stain 2: nitrilotriacetic acid, gluconic acid, sorbitol, amino alcohol, paraformaldehyde, KC1 and buffer;
Step 2, tissue section: taking a breast specimen of a subject, fixing the breast specimen by neutral formaldehyde with the concentration of 10%, taking pathological tissues, dehydrating by alcohol gradient, soaking and embedding xylene in paraffin to prepare paraffin blocks, slicing the paraffin blocks, wherein the thickness of the paraffin blocks is between 2 and 3 mu m, spreading the slices to ensure that the slices are spread sufficiently, taking out the slices by adopting glass slides treated by APES anti-drop slices, and baking the slices in an incubator at the temperature of 65 ℃ for 2 to 4 hours;
Step 3, immune refinement staining: after baking the sheet is completed, the tissue sample is fixed in a glass slide or a centrifuge tube using a formaldehyde fixing agent to maintain its morphological structure and protect the target protein, the fixed tissue sample is subjected to infiltration treatment using a penetrant, triton X-100 allows an antibody to enter cells, a pair of antibody solutions specifically binding to the target protein are added to the tissue sample, one antibody can bind to a specific epitope of the protein, the other antibody is used to detect the binding, the sample is washed with a buffer to remove unbound antibodies and other non-specifically bound substances, the signal bound to the specific antibody is amplified by using a luciferin or an enzyme substrate, the luciferin is usually observed by a fluorescent microscope, and the enzyme substrate can form a color reaction under a microscope, the tissue sample is observed using a fluorescent microscope or a normal optical microscope, and the expression of the target protein is recorded.
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