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    Absin 10-Color IHC Kit

    Absin 10-Color IHC Kit

    Catalog Number: abs50018 Brand: Absin
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    $4,320 USD
    $4,320 USD
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    Product Details

    Product Specification

    WGK Germany 0
    Usage I. Sample requirements:
    1 , formalin-fixed wax blocks or slides, large pieces of tissue or TMA The sealing wax cannot be obviously damaged.
    2 Slide samples, tissues need to be close to slides to avoid wrinkles, slides must not be damaged, scratched or stained.
    3 Tissue minimum should contain greater than 1000 Cells.
    4 Solid tumor tissues need to be embedded in wax blocks, and necrotic tumor tissues, fine needle punctures and cell slice samples will affect the staining effect.
    5 The organization should use 10% Neutral formalin fixation, normal fixation time is 18-24h 。
    6 The slice thickness is 4μm Left and right, use anti-fall slides. It is recommended that the 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 moisture, tap to remove water droplets, do not wipe the slide with paper.
    8 The glass slide is placed on 45℃ Place on a hot plate 30min (The duration of natural air-drying slides is not less than 1h ）。

    2. Inspection method:
    1 Required instruments and equipment: pipette, constant temperature drying oven, microwave oven, immunohistochemistry pen, repair cup, dyeing cylinder, timer, incubation wet box, cover glass, fume hood, washing bottle, fluorescence microscope, measuring cylinder 100mL measuring cylinder 1000mL Etc.
    2 Reagents required: sterilized deionized water, xylene, ethanol ( 100% 、 95% 、 70% ）、 10% Neutral formalin, antigen repair primary antibody, HRP labeled secondary antibodies, TBST (Item No.: abs952 ) etc.
    3 Reagent preparation:
    （ 1 ) Dilution of fluorescent dyes: all dyes were 100× Mother liquor, using chromogenic buffer 1:100 Dilute and prepare the dye working solution (prepare as it is used); TSA470SN (nuclear dyes) Use sterile water to press 1:100 Dilute the preparation working solution.
    （ 2 ) Fix / Preparation of rapid removal two-in-one working solution: 50× The concentrate was diluted with double distilled water to 1X Working fluid (powder is 16g+92mL ddH2O ）
    4 Detection equipment: fluorescence microscope or fluorescence full-film scanning equipment, TSA Excitation and emission filters suitable for monochromatic fluorescent dyes shall comply with the recommendations in the table.

    3. Paraffin section operation steps ：
    1 dewaxing and hydration
    （ 1 ) Fresh xylene dip tablets 10min , repeat 3 Twice.
    （ 2 ) Gradient ethanol dip tablets: 100% 5min ； 95% 5min ； 70% 2min 。
    （ 3 ) Sterilized water washing tablets 1min , repeat 3 Twice.
    （ 4 ） 10% Neutral formalin or 4% paraformaldehyde dip tablets 10-30min Sterilized water washing tablets 1min , repeat 3 Twice. (Optional, depending on sample quality)
    （ 5 ) Dropwise adding membrane breaking agent to permeate 15min ， TBST immersion 3min , repeat 3 Twice. (Optional, you may not do it in general)
    2 Microwave repair antigen
    （ 1 ) The dewaxed and hydrated glass slides were placed in a repair cup, and the glass slides were repaired with / Quick removal 2-in-1 working fluid immersion.
    （ 2 ) Place the repair cup in the microwave oven and bring to a high boil.
    （ 3 ) Low Fire Maintenance 15min (Pay attention to rehydration to prevent excessive evaporation causing dry tablets).
    （ 4 ) Remove to room temperature and naturally cool to room temperature.
    3 Quenching and sealing
    （ 1 ) Remove that residual lotion from the slide.
    （ 2 ) Circle the sample area on the slide with a histochemical pen and add dropwise 3% Hydrogen peroxide covers the sample area and incubates 10min ， TBST immersion 3min , repeat 3 Twice.
    （ 3 ) Remove that residual washing liquid on the slide, add the blocking liquid dropwise, cover the sample area, moisturize and shake at room temperature 10-30min The blocking solution was removed.
    4 Primary antibody incubation
    （ 1 ) Remove the blocking fluid from the slide.
    （ 2 ) The diluted primary antibody solution was added dropwise with a pipette to immerse the sample area.
    （ 3 ) Room temperature moisturizing shaking incubation 1h (It is necessary to optimize and adjust for different antibodies).
    （ 4 ) with 1×TBST buffer Immersion slide 3min , repeat 1 Twice.
    5 Secondary antibody incubation
    （ 1 ) Remove any remaining lotion from the slide.
    （ 2 ) direct dropwise addition HRP Secondary antibody working solution, immerse the sample area.
    （ 3 ) Moisturizing incubation at room temperature 10min 。
    （ 4 ) with 1×TBST buffer Immersion slide 3min , repeat 1 Twice.
    6 Fluorescence staining to amplify the signal
    （ 1 ) Remove any remaining lotion from the slide.
    （ 2 ) drop-on slide with pipette 1× Dye working solution 100μL (Use signal amplifier to press 1:100 Dilute) to immerse the sample area.
    （ 3 ) Room temperature moisturizing shaking incubation 10min 。
    （ 4 ） 1×TBST buffer Dip slides, room temperature dip slides 3min 。 repeat 3 Twice.
    （ 5 ) Microwave repair, room temperature naturally cooled to room temperature.
    （ 6 ) Sterilized water washing tablets 1 times, 1×TBST buffer Immersion tablet 2min 。
    7 A new round of dyeing (single dyeing can be directly performed on the first 9 Step)
    （ 1 ) After each round of staining, fluorescence microscope can be used to confirm the staining. TBST Cover the sample to prevent dry sheets.
    （ 2 ) Seal: Remove that residual washing liquid on the slide, drop the sealing liquid, cover the sample area, moisturize and shake at room temperature 10-30min The blocking solution was removed. (No Quenching Step Required)
    （ 3 ) Repeat steps 4-6
    （ 4 ) Multiple rounds of staining repeat steps 7 ，（ 1 ） — （ 3 Nucleation and sealing were performed after completion.
    8 Nucleation and sealing
    Dropwise 1×TSA470SN Working solution onto the sample, immerse the sample area, and incubate at room temperature 5min 。 use 1×TBST Immersion slide 3 Time, each time 2min 。 Then add antifluorescent quenching sealant dropwise, seal with cover glass, avoid bubbles, long-term storage, please use transparent nail polish to seal the edge of the cover glass.
    9 The stained tissue slices were observed and analyzed under a fluorescence microscope.

    4. Operation steps of frozen sections:
    1 Quenching, transparency, sealing
    （ 1 ) Removing the frozen glass slides from the refrigerator and restoring them to room temperature, PBST immersion 3min , repeat 3 Secondary
    （ 2 ） 10% Neutral formalin or 4% paraformaldehyde dip tablets 10-30min Sterilized water washing tablets 1min , repeat 3 Twice. (Optional, depending on sample quality)
    （ 3 ) Dropwise adding membrane breaking agent to permeate 15min ， PBST immersion 3min , repeat 3 Twice. (Optional, you may not do it in general)
    （ 4 ) Circle the sample area on the slide with a histochemical pen and add dropwise 3% Hydrogen peroxide covers the sample area and incubates 10min ， PBST immersion 3min , repeat 3 Twice.
    （ 5 ) Remove that residual washing liquid on the slide, add the blocking liquid dropwise, cover the sample area, moisturize and shake at room temperature 10-30min The blocking solution was removed.
    2 Primary antibody incubation
    （ 1 ) Remove the closure on the slide.
    （ 2 ) The diluted primary antibody solution was added dropwise with a pipette to immerse the sample area.
    （ 3 ) Room temperature moisturizing shaking incubation 1h (It is necessary to optimize and adjust for different antibodies).
    （ 4 ) with 1×TBST buffer Immersion slide 3min , repeat 1 Twice.
    3 Secondary antibody incubation
    （ 1 ) Remove any remaining lotion from the slide.
    （ 2 ) direct dropwise addition HRP Secondary antibody working solution, immerse the sample area.
    （ 3 ) Moisturizing incubation at room temperature 10min 。
    （ 4 ) with 1×TBST buffer Immersion slide 3min , repeat 1 Twice.
    4 Fluorescence staining to amplify the signal
    （ 1 ) Remove any remaining lotion from the slide.
    （ 2 ) drop-on slide with pipette 1× Components A Dye working solution 100μL (Use signal amplifier to press 1:100 Dilute) to immerse the sample area.
    （ 3 ) Room temperature moisturizing shaking incubation 10min 。
    （ 4 ） 1×TBST buffer Dip slides, room temperature dip slides 3min 。 repeat 3 Twice.
    5 、 . A new round of dyeing (single dyeing can be directly performed on the first 9 Step)
    （ 1 ) After each round of staining, fluorescence microscope can be used to confirm the staining. TBST Cover the sample to prevent dry sheets.
    （ 2 ) Seal: Remove that residual washing liquid on the slide, drop the sealing liquid, cover the sample area, moisturize and shake at room temperature 10-30min The blocking solution was removed. (No Quenching Step Required)
    （ 3 ) Repeat steps 4-6
    （ 4 ) Multiple rounds of staining repeat steps 7 ，（ 1 ） - （ 3 Nucleation and sealing were performed after completion.
    6 Nucleation and sealing
    Dropwise 1×TSA470SN Working solution onto the sample, immerse the sample area, and incubate at room temperature 5min 。 use 1×TBST Immersion slide 3 Time, each time 2min 。 Then add antifluorescent quenching sealant dropwise, seal with cover glass, avoid bubbles, long-term storage, please use transparent nail polish to seal the edge of the cover glass.
    7 、 The stained tissue slices were observed and analyzed under a fluorescence microscope.

    5. Cell climbing operation steps:
    1 Sample preparation: Prepare cell samples (cell climbing slides, cell smears, etc.). It is recommended to directly use chamber slides for cell culture to facilitate subsequent detection. Completion system After preparation, use PBS Simple rinse ( 2×2min ）。
    2 Cell fixation: with 4% Paraformaldehyde fixation at room temperature 10-20min ， PBS washing 3×5min 。
    3 Quenching endogenous peroxidase: dropwise addition 3% H2O2 , incubated at room temperature 10-30min ， PBS washing 3×5min 。
    4 Cell permeability (this step is omitted for membrane indicators): dropwise addition containing 1-0.25% Triton X-100 of PBS Solution, treated at room temperature 10min ， PBS washing 3×5min 。
    5 Blocking: dropwise adding blocking solution and incubating at room temperature 30min 。
    6 Primary antibody incubation: discard the blocking solution, drop-wise add the diluted primary antibody working solution, and place it in a light-proof wet box 4°C Incubate overnight or 37℃ 1-2h (A small amount of water can be added to the wet box to prevent antibody incubation process dry sheet), PBS Rinse 3×5min 。
    7 Secondary antibody incubation: drop addition of the species corresponding to the primary antibody HRP The tissue was covered with secondary antibody and incubated at room temperature in the dark 20-50min ， PBS Rinse 3×5min 。
    8 Dye incubation: dropwise addition of ready-prepared 1×TSA Dye working solution (using signal amplifier 1:100 Dilution), reaction 1-10min ， PBS Rinse 3×5min 。
    9 Antibody elution: antibody eluate 37℃ Preheat, shake off PBS After that, the eluent is dropwise added to infiltrate the entire climbing piece, and the elution time can be controlled at 15-30min (Difficulty of elution: structural membrane proteins and cytoskeletal proteins> cytoplasmic proteins> nuclear proteins), PBS Rinse 3×5min 。
    10 Repeating the step of performing [5→9] , until all indicator staining is completed.
    11 dropwise addition 1×TSA470SN Working solution onto the sample, immerse the sample area, and incubate at room temperature 10min ， PBS Rinse 3×5min 。
    12 Add antifluorescence quenching sealing agent dropwise, and seal the tablet with a coverslip to avoid air bubbles.

    13 , scanning imaging, data analysis.
      VI. Description of results:
    1 Changes in microwave repair antigen, incubation time and temperature may lead to wrong results.
    2 In each staining process, there must be tissue positive control and reagent negative control experiments simultaneously.
    3 If the positive tissue control cannot show positive staining, the test result of this batch of samples shall be judged to be invalid.

    VII. Product performance indicators:
    1 Conformity: Take the conforming tissue slices (including the positive tissue slices and the negative reagent control), and after the corresponding immunohistochemical test, the staining result of the positive control is positive, and the staining result of the negative control is negative.
    2 Intra-batch repeatability: Take the same batch of kits and detect the same tissue section 3 Tablet, the staining results should be consistent.
    Beilstein 0
    Experiment Reagent Instrument
    1. Reagents required: DMSO （ abs9184 ), sterilized deionized water ( abs9259 ), xylene, ethanol ( 100% 、 95% 、 70% ）、 TBST （ abs952 ) or PBST （ abs9340 ), fluorescent encapsulant ( abs9236 ）。
    2. Detection equipment: fluorescence microscope or fluorescence full film scanning equipment, PPD Excitation and emission filters suitable for the series of fluorescent dyes shall comply with the recommendations in the table below.
    Fluorescent dye name wavelength
    motivate Excitation launch Emission
    DAPI 360 461
    PPD 520 490 520
    PPD 570 550 570
    PPD 650 640 660
    Description

    There are complex cellular composition in tissue microenvironment, and the phenotype, state, abundance and distribution of these cells have important biological significance and clinical value. It can be presented in situ in tissue with the help of antibody staining. Immunohistochemical staining is a common technique to study tissue morphology and in situ protein expression. Conventional IHC detection can only display a single index, and it is difficult to present the cell composition, state and relationship in the complex tissue microenvironment, and this information is crucial for 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, and HRP catalyzes the addition of fluorescein substrate in the system to produce an activated fluorescent substrate, which can be covalently bound with tyrosine on the antigen, so that fluorescein is stable and covalently bound on the sample. Thereafter, the non-covalently bound antibody was washed off by thermal repair method, and a primary antibody was replaced for a second round of incubation, and another fluorescein substrate was replaced, so that multiple labeling can be achieved by reciprocating.
    Schematic:
    PubChem CID 0
    Wells 0
    Composition
    Component Name 50T 100T Quantity preservation
    TSA440N dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA520N dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA670N dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA570N dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA620N dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA650N dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA700N dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA540S dye 50μL 100μL 1 branch 2-8℃ Avoid light
    TSA660S dye 50μL 100μL 1 branch 2-8℃ Avoid light
    Nuclear dye 470SN 50μL 100μL 1 branch 2-8℃ Avoid light
    Rapid response buffer 45mL 45mL 1 bottle 2-8℃
    Primary antibody diluent 50mL 50mL 1 bottle 2-8℃
    Fix / Rapid removal 2-in-1 buffer 16g 16g 2 bottle 2-8℃
    3% Hydrogen peroxide 100mL 100mL 1 bottle  2-8℃
    blocking fluid 30mL 30mL 1 bottle  2-8℃
    Crystal encapsulant 10mL 10mL 1 bottle  2-8℃
    General Notes 1. This kit is only used for immunohistochemistry, not for other purposes.
    2. This kit is limited to professionals.
    3. Appropriate protective measures should be taken to avoid contact of reagents with skin and eyes.
    4. The activity of reagents that have exceeded the expiration date may be reduced, so reagents that have exceeded 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. Dewaxing is not complete, which easily affects the dyeing effect.
    7. In order to prevent possible false negative and false positive results, a positive control and a negative control should be carried out simultaneously during the experiment.
    8. All kinds of wastes generated in the use of this kit should be treated in accordance with the Regulations on Medical Waste Management.
    Application It is mainly used for the scientific research of immunohistochemical staining of tissues, paraffin sections, TMA chips, frozen sections and cell climbing slices. It can not be used for in vitro clinical diagnosis or human test.
    Storage Temp.

    2-8℃, protected from light, valid for 12 months.
    Performance Indicator

    1. Conformity: Take the conforming tissue slices (including the positive tissue slices and the negative reagent control). After the corresponding immunohistochemical test, the staining result of the positive control is positive, and the staining result of the negative control is negative.

    2. Intra-batch repeatability: Take the same batch of kits and detect 3 sections of the same tissue, and the staining results should be consistent.