Absin 7-Color IHC Kit (Anti-Rabbit&mouse Secondary Antibody),Absin_specification/price/image

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References	1. DabbsDavidJ. Diagnostic immunohistochemistry (M). Beijing: Peking University Medical Press, 2008.9. 2. [US] EdHarlow, DavidLane Antibody Technical Guide (M). Beijing: Science Press, 2002: 79-80, 105. 3. Stack, E.C. etaI., Multiplexedimmunohistochemistry, imaging, andquantitation: areview, withanassessmentofTyramidesignaIamplification, multispectraIimagingandmultiplexanalysis.Methods, 201470 （1）:46-58. 4. Qian Bangguo Jiao Lei Application of multi-label immunofluorescence staining and Doppler imaging technology in histological research. Chinese Histochemistry and Cytochemistry Journal, 2017 (4): 373-382.
Usage	Ⅰ. 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). Ⅱ. 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, blocking solution, TBST (item number: abs952), antibody diluent (item number: abs9299), etc. 3. Reagent preparation: (1) Dilution of fluorescent dyes: all dyes (including TSA monochromatic fluorescent dye 770 component A) are 100 × mother liquor, and the signal amplification reaction solution is diluted at 1: 100 to prepare the dye working solution (ready-to-use); DAPI prepares the working solution using sterile water dilution at 1: 100. (2) Dilution of TSA770 dye component B: Component B is 100 × mother liquor, and dilute 1 × PBS or antibody diluent at 1: 100 to prepare TSA770 dye component B working solution (now prepared). 4. Detection equipment: fluorescence microscope or fluorescent whole-film scanning equipment. The excitation and emission filters applicable to TSA monochromatic fluorescent dyes should comply with the recommendations in the table. Ⅲ. Paraffin sectioning operation steps 1. Dewaxing and Hydration (a) Fresh xylene dip tablets for 10 min, repeated 3 times. (b) Gradient ethanol impregnation tablets: 100% for 5 min; 95% 5 min; 70% 2 min. (c) Wash the tablet with sterilized water for 1 min and repeat it three times. (d) 10% neutral formalin or 4% paraformaldehyde dip tablets for 10-30min, sterilized water wash tablets for 1min, and repeat 3 times. (Optional, depending on sample quality) (e) Dropwise add film breaking agent to permeate for 15 min, soak with TBST for 3 min, and repeat for 3 times. (Optional, generally not necessary) 2. Microwave repair antigen (a) Place the dewaxed and hydrated glass slide in a repair cup and immerse it with antigen repair solution 1 × working solution. (b) Place the repair cup in a microwave oven and bring it to a high boil. (c) Maintain low fire for 15 minutes (pay attention to fluid rehydration to prevent excessive evaporation from causing dry tablets). (d) Remove room temperature and naturally cool to room temperature. 3. Quenching and Blocking (a) Remove that residual wash liquid on the slide. (b) Circle the sample area on the glass slide with a histochemical pen, add 3% hydrogen peroxide dropwise to cover the sample area, incubate for 10 minutes, immerse in TBST for 3 minutes, and repeat 3 times. (c) Remove that residual lotion liquid on the glass slide, add blocking liquid dropwise, cover the sample area, moisturize at room temperature and shake for 10-30min, and remove the blocking liquid. 4. Primary antibody incubation (a) Remove the blocking fluid from the slide. (b) Submerge the sample area by adding the diluted primary antibody solution dropwise with a pipette. (c) Moisturizing and shaking incubation at room temperature for 1h (optimization and adjustment need to be made for different antibodies). (d) The slides were dipped with 1 × TBSTbuffer for 3 min and repeated once. 5. Secondary antibody incubation (a) Remove the residual wash solution on the slide. (b) Directly add HRP secondary antibody working solution dropwise to immerse the sample area. (c) Incubate at room temperature with moisture for 10 min. (d) The slides were dipped with 1 × TBSTbuffer for 3 min and repeated once. 6. Fluorescence staining amplifies signal (a) Remove the residual wash solution on the slide. (b) Immerse the sample area by adding 1 × 100 ul of dye working solution dropwise on the glass slide with a pipette (diluted 1: 100 with signal amplification solution). (c) Incubate at room temperature with moisture shaking for 10 min. (d) 1 × TBSTbuffer dip slides and dip them at room temperature for 3 min. Repeat 3 times. (e) Microwave repair, natural cooling at room temperature to room temperature. (f) Wash the tablets once with sterilized water and dip the tablets in 1 × TBSTbuffer for 2 minutes. 7. A new round of dyeing (single dyeing can be carried out directly to step 9) (a) 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. (b) Blocking: Remove the residual lotion solution 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. (No quenching step required) (c) Repeat steps 4-6 (d) Repeat step 7 for multiple rounds of dyeing, and after completion of a)-c), dyeing nuclei and sealing are carried out. (Last round staining 770 fluorescent dye component A) 8. TSA770 Dye Component B Add 50-100ul 1 × TSA770 component B working solution dropwise, immerse the sample area, and incubate at room temperature for 10-30min (the staining time should be judged according to the staining results). The slides were dipped 3 times with 1 × TBST for 3 minutes each time. 9. Dyeing and sealing Add 1 × DAPI 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. 10. Read the film, observe and analyze the stained tissue slice under a fluorescence microscope. Ⅳ. Frozen section operation steps (antibody eluate abs994 is required) 1. Quenching, permeation, sealing (a) Remove the frozen slides from the refrigerator, return to room temperature, soak in PBST for 3 min, and repeat 3 times (b) 10% neutral formalin or 4% paraformaldehyde dipping tablets for 10-30min, washing tablets with sterilized water for 1min, repeating 3 times. (Optional, depending on sample quality) (c) Dropwise add membrane breaking agent for permeation for 15 min, soak PBST for 3 min, and repeat 3 times. (Optional, generally not necessary) (d) Circle the sample area on the glass slide with a histochemical pen, add 3% hydrogen peroxide dropwise to cover the sample area, incubate for 10 minutes, soak with PBST for 3 minutes, and repeat 3 times. (e) Remove that residual lotion liquid on the glass slide, add blocking liquid dropwise, cover the sample area, moisturize at room temperature and shake for 10-30min, and remove the blocking liquid. 2. Primary antibody incubation (a) Remove the closure on the slide. (b) Submerge the sample area by adding the diluted primary antibody solution dropwise with a pipette. (c) Moisturizing and shaking incubation at room temperature for 1h (optimization and adjustment need to be made for different antibodies). (d) The slides were dipped with 1 × TBSTbuffer for 3 min and repeated once. 3. Secondary antibody incubation (a) Remove the residual wash solution on the slide. (b) Directly add HRP secondary antibody working solution dropwise to immerse the sample area. (c) Incubate at room temperature with moisture for 10 min. (d) The slides were dipped with 1 × TBSTbuffer for 3 min and repeated once. 4. Fluorescence staining amplification signal (a) Remove the residual wash solution on the slide. (b) Immerse the sample area by adding 1 × 100 ul of component A dye working solution dropwise on the glass slide with a pipette (diluted 1: 100 with signal amplification solution). (c) Incubate at room temperature with moisture shaking for 10 min. (d) 1 × TBSTbuffer dip slides and dip them at room temperature for 3 min. Repeat 3 times. 5. A new round of dyeing (single dyeing can be carried out directly to step 9) (a) 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. (b) Blocking: Remove the residual lotion solution 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. (No quenching step) c) Repeat steps 4-6 (d) Repeat step 7 for multiple rounds of dyeing, and after completion of a)-c), dyeing nuclei and sealing are carried out. (Last round staining 770 fluorescent dye component A) 6. TSA770 Dye Component B Add 50-100ul 1 × TSA770 component B working solution dropwise, immerse the sample area, and incubate at room temperature for 10-30min (the staining time should be judged according to the staining results). The slides were dipped 3 times with 1 × TBST for 3 minutes each time. 7. Dyeing and sealing Add 1 × DAPI 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. 8. Read the film, observe and analyze the stained tissue slice under a fluorescence microscope. Ⅴ. 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. Ⅵ. 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.
Synonym	Multicolor kit
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. This information is crucial to the diagnosis and treatment of diseases! Principle of tyrosine signal amplification technology: Similar to the DAB color development method of conventional immunohistochemistry, TSA technology also uses HRP-labeled secondary antibody. HRP catalyzes the fluorescein substrate added to the system to produce an activated fluorescein substrate, which can be covalently bound to the antigen. Tyrosine is covalently bound to stably covalently bound to fluorescein on the sample. After that, the non-covalently bound antibody is washed away by thermal repair, replaced with a primary antibody for the second round of incubation, and replaced with another fluorescein substrate, so that multiple labeling can be achieved by reciprocating.
Composition	Kit Components TSA monochromatic fluorescent dye 480, TSA monochromatic fluorescent dye 520, TSA monochromatic fluorescent dye 570, TSA monochromatic fluorescent dye 620, TSA monochromatic fluorescent dye 690, TSA monochromatic fluorescent dye 770 component A, TSA770 dye component B, signal amplification reaction solution, anti-pika universal HRP labeled secondary antibody, anti-fluorescence quenching sealing tablet, DAPI
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.
Storage Temp.	Fluorescent dyes should be stored in the dark from light at 2-8 ℃, and the validity period is 12 months from receipt of the goods.

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.