Absin 10-Color IHC Kit
Absin 10-Color IHC Kit
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Product Specification
| Usage |
1. Sample requirements: 1. Formalin-fixed wax blocks or glass slides, large tissues or TMA, and sealing wax cannot be obviously damaged. 2. For glass slide samples, the tissue needs to be close to the glass slide to avoid wrinkles, and the glass slide must not be damaged, scratched or stained. 3. The tissue should contain at least more than 1000 cells. 4. The wax block needs to be embedded with solid tumor tissue, and necrotic tumor tissue, fine needle punctures, and cell slice samples will affect the staining effect. 5. The tissue should be fixed with 10% neutral formalin, and the normal fixation time is 18-24h. 6. The thickness of the slice is about 4um, and anti-detachment slides are used. It is recommended that the glass slides be prepared within one week after fixation. 7. Do not add any adhesive in the water bath fishing process. The sample should be placed on the front and center of the slide. Place the slide vertically on absorbent paper to remove water, tap gently to remove water droplets, and do not wipe the slide with paper. 8. Place the glass slide on a hot plate at 45 °C for 30 minutes (the natural air-drying time of the glass slide is not less than 1h). 2. Inspection method: 1. Required instruments and equipment: pipette, constant temperature drying oven, microwave oven, immunohistochemical pen, repair cup, dyeing cylinder, timer, incubation wet box, cover glass, fume hood, bottle washing, fluorescence microscope, measuring cylinder 100mL, measuring cylinder 1000mL, etc. 2. Required reagents: sterilized deionized water, xylene, ethanol (100%, 95%, 70%), 10% neutral formalin, antigen repair primary antibody, HRP labeled secondary antibody, TBST (item number: abs952) etc. 3. Reagent preparation: (1) Dilution of fluorescent dyes: all dyes are 100 × mother liquor, and the color buffer is diluted at 1: 100 to prepare the dye working solution (ready-to-use); TSA470SN (core dye) is used to prepare the working solution with sterile water dilution at 1: 100. (2) Preparation of repair/quick removal two-in-one working solution: 50 × concentrated solution is diluted with double distilled water to 1X working solution (powder is 16g + 92mL ddH2O) 4. Detection equipment: fluorescence microscope or fluorescence whole-film scanning equipment. The excitation and emission filters applicable to TSA monochromatic fluorescent dyes should comply with the recommendations in the table. 3. Paraffin sectioning operation steps： 1. Dewaxing and hydration (1) Dip fresh xylene tablets for 10 minutes and repeat 3 times. (2) Gradient ethanol dipping tablets: 100% for 5min; 95% 5 min; 70% 2 min. (3) Wash the tablets with sterilized water for 1min and repeat 3 times. (4) Dip the tablets in 10% neutral formalin or 4% paraformaldehyde for 10-30min, wash the tablets with sterilized water for 1min, and repeat 3 times. (Optional, depending on sample quality) (5) Add film breaking agent dropwise for 15 minutes, soak in TBST for 3 minutes, and repeat 3 times. (Optional, generally not necessary) 2. Microwave repair antigen (1) Place the dewaxed and hydrated glass slide in the repair cup and immerse it with the repair/quick removal two-in-one working fluid. (2) Place the repair cup in the microwave oven and bring it to a high boil. (3) Maintain low fire for 15 minutes (pay attention to rehydration to prevent excessive evaporation from leading to dry tablets). (4) Take out room temperature and naturally cool to room temperature. 3. Quenching and blocking (1) Remove the residual lotion solution on the glass slide. (2) Circle the sample area on the glass slide with a histochemical pen, add 3% hydrogen peroxide dropwise to cover the sample area, incubate for 10min, soak in TBST for 3min, and repeat 3 times. (3) Remove the residual lotion on the glass slide, add the blocking solution dropwise, cover the sample area, moisturize at room temperature and shake for 10-30min, and remove the blocking solution. 4. Primary antibody incubation (1) Remove the blocking fluid on the slide. (2) Add the diluted primary antibody solution dropwise with a pipette to immerse the sample area. (3) Moisturizing and shaking at room temperature for 1h (optimization and adjustment need to be made for different antibodies). (4) Dip the glass slide with 1 × TBST buffer for 3 minutes and repeat once. 5. Secondary antibody incubation (1) Remove the lotion remaining on the glass slide. (2) Directly add HRP secondary antibody working solution dropwise to immerse the sample area. (3) Moisturizing at room temperature and incubating for 10min. (4) Dip the glass slide with 1 × TBST buffer for 3 minutes and repeat once. 6. Fluorescence staining amplifies signal (1) Remove the lotion remaining on the glass slide. (2) Use a pipette to dropwise add 1 × 100ul of dye working solution on the glass slide (dilute 1: 100 with signal amplification solution) to immerse the sample area. (3) Moisturizing at room temperature and shaking incubation for 10min. (4) Dip the glass slides with 1 × TBST buffer and dip the slides at room temperature for 3 minutes. Repeat 3 times. (5) Microwave repair, natural cooling at room temperature to room temperature. (6) Wash the tablets with sterilized water once, and dip the tablets in 1 × TBST buffer for 2min. 7. A new round of dyeing (single dyeing can be carried out directly to step 9) (1) After each round of staining, a fluorescence microscope can be used to confirm the staining. Pay attention to covering the sample with TBST to prevent drying. (2) Sealing: Remove the residual lotion solution on the glass slide, add the sealing solution dropwise, cover the sample area, moisturize at room temperature and shake for 10-30min, and remove the sealing solution. (No quenching step required) (3) Repeat steps 4-6 (4) After multiple rounds of dyeing are repeated in step 7, a)-c), dyeing nuclei and sealing are carried out. 8. Nucleation and sealing Add 1 × TSA470SN working solution dropwise to the sample, immerse the sample area, and incubate at room temperature for 5 minutes. The slides were dipped 3 times with 1 × TBST for 2 minutes each time. Then add anti-fluorescence quenching sealing tablets dropwise, and seal the tablets with coverslips to avoid bubbles. Please seal the edges of coverslips with transparent nail polish for long-term storage. 9. Read the film, observe and analyze the stained tissue slice under a fluorescence microscope. 4. Frozen section operation steps: 1. Quenching, permeability, sealing (1) Take out the frozen glass slide from the refrigerator, restore it to room temperature, soak it in PBST for 3min, and repeat 3 times (2) Dip the tablets in 10% neutral formalin or 4% paraformaldehyde for 10-30min, wash the tablets with sterilized water for 1min, and repeat 3 times. (Optional, depending on sample quality) (3) Add film breaking agent dropwise for 15 minutes, soak with PBST for 3 minutes, and repeat 3 times. (Optional, generally not necessary) (4) Circle the sample area on the glass slide with a histochemical pen, add 3% hydrogen peroxide dropwise to cover the sample area, incubate for 10min, soak in PBST for 3min, and repeat 3 times. (5) Remove the residual lotion on the glass slide, add the blocking solution dropwise, cover the sample area, moisturize at room temperature and shake for 10-30min, and remove the blocking solution. 2. Primary antibody incubation (1) Remove the closure on the slide. (2) Add the diluted primary antibody solution dropwise with a pipette to immerse the sample area. (3) Moisturizing and shaking at room temperature for 1h (optimization and adjustment need to be made for different antibodies). (4) Dip the glass slide with 1 × TBST buffer for 3 minutes and repeat once. 3. Secondary antibody incubation (1) Remove the lotion remaining on the glass slide. (2) Directly add HRP secondary antibody working solution dropwise to immerse the sample area. (3) Moisturizing at room temperature and incubating for 10min. (4) Dip the glass slide with 1 × TBST buffer for 3 minutes and repeat once. 4. Fluorescence staining amplifies signal (1) Remove the lotion remaining on the glass slide. (2) Use a pipette to dropwise add 1 × Component A dye working solution 100ul on the glass slide (dilute 1: 100 with signal amplification solution) to immerse the sample area. (3) Moisturizing at room temperature and shaking incubation for 10min. (4) Dip the glass slides with 1 × TBST buffer and dip the slides at room temperature for 3 minutes. Repeat 3 times. 5. A new round of dyeing (single dyeing can be carried out directly to step 9) (1) After each round of staining, a fluorescence microscope can be used to confirm the staining. Pay attention to covering the sample with TBST to prevent drying. (2) Sealing: Remove the residual lotion solution on the glass slide, add the sealing solution dropwise, cover the sample area, moisturize at room temperature and shake for 10-30min, and remove the sealing solution. (No quenching step required) (3) Repeat steps 4-6 (4) Repeat step 7 for multiple rounds of dyeing, and carry out core dyeing and film sealing after (1)-(3) are completed. 6. Nucleation and sealing Add 1 × TSA470SN working solution dropwise to the sample, immerse the sample area, and incubate at room temperature for 5 minutes. The slides were dipped 3 times with 1 × TBST for 2 minutes each time. Then add anti-fluorescence quenching sealing tablets dropwise, and seal the tablets with coverslips to avoid bubbles. Please seal the edges of coverslips with transparent nail polish for long-term storage. 7. Read the film, observe and analyze the stained tissue slice under a fluorescence microscope. 5. Cell climbing operation steps: 1. Sample preparation: To prepare cell samples (cell slides, cell smears, etc.), it is recommended to directly use chamber slides for cell culture to facilitate subsequent detection. Upon completion of the preparation, it was briefly rinsed with PBS (2 × 2 min). 2. Cell fixation: Fixed with 4% paraformaldehyde at room temperature for 10-20min, and washed with PBS for 3 × 5min. 3. Quench the endogenous peroxidase: add 3% H2O2 dropwise, incubate at room temperature for 10-30min, and wash with PBS for 3 × 5min. 4. Cell permeability (this step is omitted for cell membrane index): Add PBS solution containing 1-0.25% Triton X-100 dropwise, treat at room temperature for 10 minutes, and wash with PBS for 3 × 5 minutes. 5. Blocking: Add blocking solution dropwise and incubate at room temperature for 30 minutes. 6. Primary antibody incubation: Discard the blocking solution, add the diluted primary antibody working solution dropwise, and incubate in a dark wet box at 4 °C overnight or 37 °C for 1-2h (a small amount of water can be added to the wet box to prevent the antibody from drying out during incubation), rinse with PBS for 3 × 5 minutes. 7. Secondary antibody incubation: Dropwise add HRP secondary antibody of the corresponding species to the primary antibody to cover the tissue, incubate at room temperature in the dark for 20-50min, and rinse with PBS for 3 × 5min. 8. Dye incubation: Dropwise add the freshly prepared 1 * TSA dye working solution (dilute 1: 100 with signal amplification solution), react for 1-10 minutes, and rinse with PBS for 3 × 5 minutes. 9. Antibody elution: Preheat the antibody eluate at 37 °C. After removing the PBS, add the eluate dropwise to infiltrate the entire crawling slice. The elution time can be controlled at 15-30min (elution difficulty: structural membrane protein and cytoskeletal protein > cytoplasmic protein > nuclear protein), rinse with PBS for 3 × 5 min. 10. Repeat step [5 → 9] until all indicator staining is completed. 11. Add 1 * TSA470SN working solution dropwise to the sample, immerse the sample area, incubate at room temperature for 10 minutes, and rinse with PBS for 3 × 5 minutes. 12. Add anti-fluorescence quenching sealing tablets dropwise and seal them with coverslips to avoid bubbles. 13. Scanning imaging and data analysis. VI. Description of results: 1. The changes of microwave repair antigen, incubation time and temperature may all lead to wrong results. 2. In each staining process, tissue positive control and reagent negative control experiments must be carried out at the same time. 3. If the positive tissue control cannot show positive staining, the test results of this batch of samples should be judged to be invalid. 7. Product performance indicators: 1. Conformity: Take the conforming tissue slice (including positive tissue slice and negative reagent control), and after the corresponding immunohistochemical test, the positive control staining result is positive, and the negative control staining result is negative. 2. Intra-batch repeatability: Take the same batch of kits and detect 3 sections of the same tissue. The staining results should be consistent. |
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| Description | There are complex cells in tissue microenvironment, and the phenotype, state, abundance and distribution of these cells have important biological significance and clinical value. It can be rendered in situ in tissue by means of antibody staining. Immunohistochemical staining is a common technique to study tissue morphology and protein expression in situ. Routine IHC detection can only show a single indicator, and it is difficult to present the cell composition, state and relationship in the complex tissue microenvironment, and this information is crucial to the diagnosis and treatment of diseases. |
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| Composition |
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| General Notes | 1. This kit is only used for immunohistochemistry and not for other purposes. 2. This kit is limited to professional use. 3. Appropriate protective measures should be taken to avoid contact between the reagent and the skin and eyes. 4. The activity of reagents beyond the expiration date may be reduced, so reagents beyond the expiration date should not be used. 5. If the dyeing components of this kit are mixed with products of other companies, abnormalities may occur during the dyeing process. 6. Incomplete dewaxing will easily affect the dyeing effect. 7. In order to prevent possible false negative and false positive results, positive control and negative control should be carried out at the same time during the experiment. 8. All kinds of wastes generated in the use of this kit should be disposed of in accordance with the Regulations on Medical Waste Management. |
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| Application | It is mainly used for scientific research on immunohistochemical staining of tissues, paraffin sections, TMA chips, frozen sections and cell crawling slices, and cannot be used for in vitro clinical diagnosis or human experiments. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Storage Temp. | 2-8 ℃, protected from light, valid for 12 months. |
