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    mIHC Dye (TSA520)

    mIHC Dye (TSA520)

    Catalog Number: abs50076 Brand: Absin
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    $450 USD
    $450 USD
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    Product Details

    Product Specification

    References 1. Dabbs David J. Diagnostic immunohistochemistry (M). Beijing: Peking University Medical Press, 2008.9. 2. [US] Ed Harlow, David Lane Technical Guide (M). Beijing: Science Press, 2002: 79-80, 105. Stack, E.C., et al., Multiplexed immunohistochemistry, imaging, and uantitation: are view, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods, 2014; 70 (1): 46-58. 4. Qian Bangguo Jiao Lei Application of multi-label immunofluorescence staining and Doppler imaging technology in histological research. Chinese Journal of Histochemistry and Cytochemistry, 2017 (4): 373-382.
    Usage I. Sample Requirements
     1. Formalin-fixed paraffin blocks or slides; large tissue blocks or TMAs; paraffin seal must be intact.
    2. For slide samples, tissue must adhere tightly to the slide without folds; slides must be free of cracks, scratches or stains.
    3. Tissue must contain >1,000 cells.
    4. Only solid-tumor tissue should be embedded; necrotic tumor, fine-needle aspirates or cytospins will impair staining.
    5. Tissue must be fixed in 10 % neutral buffered formalin for 18–24 h.
    6. Cut 4-µm sections on adhesion slides; prepare slides within one week after fixation.
    7. Do NOT add any adhesives to the water bath; place tissue on the front centre of the slide. Remove water by standing the slide vertically on absorbent paper; tap off drops—never wipe.
    8. Dry slide on a 45 °C hot-plate for 30 min (air-dry ≥1 h if no hot-plate).

    II. Assay Procedure
     1. Equipment required: pipettes, oven, microwave, IHC pen, retrieval jar, staining jars, timer, humid chamber, cover-slips, fume hood, wash bottle, fluorescence microscope, 100 mL & 1,000 mL cylinders, etc.
    2. Reagents required: sterile water (abs9259), xylene, ethanol (100 %, 95 %, 70 %), 10 % neutral buffered formalin, antigen-retrieval buffer, primary antibodies, polymer HRP-secondary antibodies (abs20216, abs50213), blocking buffer, TBST, anti-fade mounting medium.
    3. Reagent preparation:
       1) Fluorophore dilution: 100× stock dyes are diluted 1:100 in amplification buffer (prepare fresh).
    4. Imaging: fluorescence microscope or slide scanner; filter sets must match dye specifications below.

    III. Paraffin-Section Protocol
     1. De-paraffinisation & re-hydration
    a) Fresh xylene 10 min ×3.
    b) Ethanol gradient: 100 % 5 min → 95 % 5 min → 70 % 2 min.
    c) Sterile water 1 min ×3.
    d) Optional: 10 % neutral formalin or 4 % PFA 10–30 min, then water 1 min ×3.
    e) Optional: permeabilisation 15 min, PBST 3 min ×3.
    2. Antigen retrieval (microwave)
    a) Place slides in retrieval buffer (1×) in microwave jar.
    b) High power to boil; maintain low boil 15 min (top up liquid).
    c) Cool to RT.
    3. Quench & block
    a) Remove liquid; circle tissue with IHC pen.
    b) 3 % H₂O₂ 10 min; PBST 3 min ×3.
    c) Blocking buffer 10–30 min RT with shaking; discard.
    4. Primary antibody
    a) Add diluted primary Ab to cover tissue.
    b) Incubate 1 h RT with shaking; optimise if needed.
    c) TBST 3 min ×2.
    5. Secondary antibody
    a) Polymer-HRP secondary (abs20216 or abs50213) 10 min RT.
    b) TBST 3 min ×2.
    6. Fluorescent TSA amplification
    a) Apply 100 µL 1× dye working solution 10 min RT with shaking.
    b) TBST 3 min ×3.
    c) Microwave retrieval; cool to RT.
    d) Sterile water 1 min → TBST 2 min.
    7. Next cycle (skip for single stain)
    a) Check signal under microscope (keep tissue wet).
    b) Block 10–30 min (no H₂O₂ quench).
    c) Repeat steps 4–6.
    d) After final cycle proceed to nuclear staining.
    8. Nuclear stain & mounting
    a) 1× DAPI 5 min RT.
    b) TBST 2 min ×3.
    c) Add anti-fade medium; cover-slip, avoid bubbles; seal edges with clear nail polish for long-term storage.
    9. Image & analyse.

    IV. Frozen-Section Protocol (requires antibody-elution buffer abs994)
     1. Quench, permeabilise, block
    a) Equilibrate slide to RT; PBST 3 min ×3.
    b) Optional: 10 % neutral formalin or 4 % PFA 10–30 min; water 1 min ×3.
    c) Optional: permeabilisation 15 min; PBST 3 min ×3.
    d) 3 % H₂O₂ 10 min; PBST 3 min ×3.
    e) Blocking buffer 10–30 min RT; discard.
    2. Primary antibody – same as paraffin step 4.
    3. Secondary antibody – same as paraffin step 5.
    4. TSA amplification
    a) 1× dye working solution 10 min RT.
    b) TBST 3 min ×3.
    c) Antibody-elution buffer (abs994) 15–20 min 37 °C.
    d) Sterile water 1 min → TBST 2 min.
    5. Next cycle (skip for single stain)
    a) Check signal; keep wet.
    b) Block 10–30 min.
    c) Repeat steps 2–4.
    6. Nuclear stain & mounting – identical to paraffin step 8.
    7. Image & analyse.

    V. Result Interpretation
     1. Any change in retrieval, incubation time or temperature may invalidate results.
    2. Each run must include positive tissue control and negative reagent control.
    3. If positive control fails, the entire batch is invalid.

    VI. Performance Characteristics
     1. Accuracy: positive control tissue shows expected staining; negative control is blank.
    2. Within-batch precision: triplicate sections stained with the same kit lot must yield identical patterns.
    Multiplex fluorescent IHC demonstration video

    VII. Dye Spectral Data
    Dye Wavelength (nm) Analogues Display Colour
    Ex Em
    DAPI 360 461 DAPI           
    TSA 480 450 480 Opal480/Aqua           
    TSA 520 490 520 FITC/AF488/Opal520           
    TSA 540 520 540 AF514/Opal540           
    TSA 570 550 570 Cy3/AF555/Opal570           
    TSA 620 590 620 AF594/Opal620/Texas-Red           
    TSA 650 630 650 Cy5/AF647/Opal650           
    TSA 690 640 670 AF647/Cy5/Opal670           
    TSA 700 680 702 AF680/Cy5.5/Opal690           
    TSA 770 740 780 Cy7/Opal780           

    VIII. Related Products

    Category Cat. No. Product Name Size
    Fixation abs9179 4 % Paraformaldehyde (universal fixative) 500 mL
    Embedding (frozen) abs9756 OCT Compound 110 mL
    Antigen retrieval (paraffin) abs9342 Tris-EDTA retrieval buffer (10×, pH 9.0) 100 mL
    abs9248 Citrate retrieval buffer (50×) 100 mL
    Antibody elution (cells, frozen, bone) abs994 Antibody Elution Buffer (mIHC grade) 30 mL
    Permeabilisation abs9149 Triton X-100 100 mL
    Endogenous peroxidase quench abs9333 Peroxidase Block (H₂O₂ method) 100 mL
    abs9152 Tween-20 500 mL / 2.5 L
    Blocking (serum) abs933 Goat Serum 500 mL
    abs9157 BSA 100 g
    Primary antibodies abs1 IHC-validated antibodies —
    abs9299 Ab diluent 100 mL
    Multiplex kits (TSA dyes, DAPI, mount, secondary, buffer) abs50165 7-Color mIHC Kit (770 enhanced, anti-rabbit) 20 T / 50 T / 100 T
    abs50166 7-Color mIHC Kit (770 enhanced, universal) 20 T / 50 T / 100 T
    abs50015 7-Color mIHC Kit (universal) 20 T / 50 T / 100 T
    abs50037 7-Color mIHC Kit (plus, universal) 20 T / 50 T / 100 T
    abs50031 7-Color mIHC Kit (anti-rabbit) 20 T / 50 T / 100 T
    abs50038 7-Color mIHC Kit (plus, anti-rabbit) 20 T / 50 T / 100 T
    abs50014 6-Color mIHC Kit (universal) 20 T / 50 T / 100 T
    abs50049 6-Color mIHC Kit (plus, universal) 20 T / 50 T / 100 T
    abs50030 6-Color mIHC Kit (anti-rabbit) 20 T / 50 T / 100 T
    abs50048 6-Color mIHC Kit (plus, anti-rabbit) 20 T / 50 T / 100 T
    abs50013 5-Color mIHC Kit (universal) 20 T / 50 T / 100 T
    abs50029 5-Color mIHC Kit (anti-rabbit) 20 T / 50 T / 100 T
    abs50012 4-Color mIHC Kit (universal) 20 T / 50 T / 100 T
    abs50028 4-Color mIHC Kit (anti-rabbit) 20 T / 50 T / 100 T
    abs50089 3-Color mIHC Kit (universal) 100 T
    abs50088 3-Color mIHC Kit (anti-rabbit) 100 T
    abs50087 2-Color mIHC Kit (universal) 100 T
    abs50086 2-Color mIHC Kit (anti-rabbit) 100 T
    Lung-cancer panels (include primaries) abs50083 Lung TME mIHC Panel I 20 T
    abs50084 Lung TME mIHC Panel II 20 T
    Wash buffers abs952 TBST (10×) 500 mL ×2
    abs9340 PBST (1×, pH 7.4) 500 mL
    abs962 PBS (1×) 500 mL
    Polymer-HRP secondary abs20216 High-sensitivity Poly-HRP Goat anti-Rabbit IgG (H+L) 100 mL
    abs50213 High-sensitivity Poly-HRP Goat anti-Rabbit/Mouse IgG (H+L) 100 mL
    Theory Multiplex fluorescence immunohistochemical staining is based on the principle of specific binding of antigen and antibody. The secondary antibody labeled with horseradish peroxidase (HRP) is selected to activate the fluorescent dye in the kit and covalently bind the signal to the antigen. In situ multi-target staining of tissues or cells is achieved by multiple rounds of staining cycles with the aid of different fluorescent dye labels.
    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 method, 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 TSA monochromatic fluorescent dye 520, signal amplification reaction solution
    General Notes 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 in 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 slide 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 30min (the natural air-drying time of the glass slide is not less than 1h).
    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.
    Applications It is mainly used for scientific research on immunohistochemical staining of tissues, paraffin sections and TMA chips, and cannot be used for in vitro clinical diagnosis or human experiments.