mIHC Dye (TSA540)
mIHC Dye (TSA540)
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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
II. Assay Method1. Required EquipmentMicropipettes, constant-temperature drying oven, microwave oven, immunohistochemistry (IHC) hydrophobic barrier pen, antigen retrieval container, staining jars, timer, humidified incubation chamber, coverslips, fume hood, wash bottles, fluorescence microscope, 100 mL and 1,000 mL graduated cylinders, etc.
2. Required ReagentsSterile deionized water (abs9259), xylene, ethanol (100%, 95%, 70%), 10% neutral buffered formalin, antigen retrieval solution, primary antibodies, polymer HRP-conjugated secondary antibodies (abs20216, abs50213), blocking solution, PBST, anti-fade mounting medium, etc.
3. Reagent Preparation
4. Detection EquipmentFluorescence microscope or whole-slide fluorescence scanning system. Excitation and emission filter sets for TSA monochromatic fluorophores should conform to the specifications listed in Table 7.
III. Protocol for FFPE Tissue Sections1. Deparaffinization and Rehydration(1) Immerse slides in fresh xylene for 10 min, repeat 3 times.
(2) Gradient ethanol rehydration: 100% ethanol for 5 min; 95% ethanol for 5 min; 70% ethanol for 2 min. (3) Rinse in sterile water for 1 min, repeat 3 times. (4) Optional: Immerse in 10% NBF or 4% paraformaldehyde (PFA) for 10–30 min, followed by sterile water rinse for 1 min, repeat 3 times (perform based on sample quality). (5) Optional: Apply membrane permeabilization reagent for 15 min, wash in PBST for 3 min, repeat 3 times (generally not required). 2. Microwave Antigen Retrieval(1) Place deparaffinized slides in an antigen retrieval container and submerge in 1× antigen retrieval working solution.
(2) Heat in a microwave at high power until boiling. (3) Maintain at low power for 15 min (replenish buffer as needed to prevent tissue desiccation). (4) Remove and allow to cool naturally to room temperature. 3. Quenching and Blocking(1) Remove residual wash buffer from slides.
(2) Encircle the specimen area with an IHC hydrophobic barrier pen. Apply peroxidase blocking solution to cover the specimen area, incubate for 10 min, then wash in PBST for 3 min, repeat 3 times. (3) Remove residual wash buffer. Apply blocking solution to cover the specimen area, incubate at room temperature for 60 min, then remove blocking solution. 4. Primary Antibody Incubation(1) Remove blocking solution from slides.
(2) Apply diluted primary antibody solution to cover the specimen area using a micropipette. (3) Incubate in a humidified chamber with gentle agitation at room temperature for 1 h (optimize for individual antibodies) or overnight at 4°C. (4) Wash in 1× PBST for 3 min, repeat 3 times. 5. Secondary Antibody Incubation(1) Remove residual wash buffer from slides.
(2) Apply polymer HRP-conjugated secondary antibody working solution to cover the specimen area. (3) Incubate in a humidified chamber at room temperature for 30 min. (4) Wash in 1× PBST for 3 min, repeat 3 times. 6. Fluorescent Signal Amplification(1) Remove residual wash buffer from slides.
(2) Apply 50–100 µL of 1× fluorophore working solution (diluted 1:200 in signal amplification buffer) to cover the specimen area. (3) Incubate in a humidified chamber with gentle agitation at room temperature for 10–15 min. (4) Wash in 1× PBST at room temperature for 3 min, repeat 3 times. 7. Subsequent Rounds of Staining (Proceed directly to Step 8 for single staining)(1) After each round, staining quality may be verified under a fluorescence microscope. Ensure slides are kept moist with PBST to prevent desiccation.
(2) Elution: Remove residual wash buffer. Apply antibody elution buffer to cover the specimen area, incubate at 37°C for 20–30 min, then remove elution buffer (no additional quenching step required). (3) Repeat Steps 4–6. (4) For multiplex staining, repeat Step 7(1)–(3), then proceed to nuclear counterstaining and mounting. 8. Nuclear Counterstaining and MountingApply 1× DAPI working solution to cover the specimen area, incubate at room temperature for 10 min. Wash in 1× PBST 3 times, 2 min each. Apply anti-fade mounting medium and cover with a coverslip, avoiding air bubbles. For long-term storage, seal the edges of the coverslip with clear nail polish.
9. Image Acquisition and AnalysisObserve and analyze stained tissue sections under a fluorescence microscope.
IV. Protocol for Frozen Tissue Sections (Requires Antibody Elution Buffer abs994)1. Quenching, Permeabilization, and Blocking(1) Remove frozen slides from the freezer, equilibrate to room temperature, wash in PBST for 3 min, repeat 3 times.
(2) Optional: Immerse in 10% NBF or 4% PFA for 10–30 min, followed by sterile water rinse for 1 min, repeat 3 times (perform based on sample quality). (3) Optional: Apply membrane permeabilization reagent for 15 min, wash in PBST for 3 min, repeat 3 times (generally not required). (4) Encircle the specimen area with an IHC hydrophobic barrier pen. Apply peroxidase blocking solution to cover the specimen area, incubate for 10 min, then wash in PBST for 3 min, repeat 3 times. (5) Remove residual wash buffer. Apply blocking solution to cover the specimen area, incubate at room temperature for 60 min, then remove blocking solution. 2. Primary Antibody Incubation(1) Remove blocking solution from slides.
(2) Apply diluted primary antibody solution to cover the specimen area using a micropipette. (3) Incubate in a humidified chamber with gentle agitation at room temperature for 1 h (optimize for individual antibodies) or overnight at 4°C. (4) Wash in 1× PBST for 3 min, repeat 3 times. 3. Secondary Antibody Incubation(1) Remove residual wash buffer from slides.
(2) Apply HRP-conjugated secondary antibody working solution to cover the specimen area. (3) Incubate in a humidified chamber at room temperature for 30 min. (4) Wash in 1× PBST for 3 min, repeat 3 times. 4. Fluorescent Signal Amplification(1) Remove residual wash buffer from slides.
(2) Apply 50–100 µL of 1× fluorophore working solution (diluted 1:200 in signal amplification buffer) to cover the specimen area. (3) Incubate in a humidified chamber with gentle agitation at room temperature for 10 min. (4) Wash in 1× PBST at room temperature for 3 min, repeat 3 times. (5) Apply antibody elution buffer (abs994), incubate at 37°C for 15–20 min. (6) Rinse in sterile water once, then wash in 1× PBST for 2 min. 5. Subsequent Rounds of Staining (Proceed directly to Step 6 for single staining)(1) After each round, staining quality may be verified under a fluorescence microscope. Ensure slides are kept moist with PBST to prevent desiccation.
(2) Repeat Steps 2–4. (3) For multiplex staining, repeat Step 5, then proceed to nuclear counterstaining and mounting. 6. Nuclear Counterstaining and MountingApply 1× DAPI working solution to cover the specimen area, incubate at room temperature for 10 min. Wash in 1× PBST 3 times, 2 min each. Apply anti-fade mounting medium and cover with a coverslip, avoiding air bubbles. For long-term storage, seal the edges of the coverslip with clear nail polish.
7. Image Acquisition and AnalysisObserve and analyze stained tissue sections under a fluorescence microscope.
V. Interpretation of Results
VI. Product Performance Specifications
VII. Fluorophore Information
VIII. Recommended Companion Products
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| 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 |
The tissue microenvironment comprises a complex array of cellular constituents, whose phenotypes, functional states, abundances, and spatial distributions hold profound biological significance and clinical relevance. These parameters can be visualized in situ through antibody-based staining methodologies. While conventional immunohistochemistry (IHC) represents a standard approach for investigating tissue morphology and in situ protein expression, traditional single-marker IHC is inherently limited in its ability to elucidate the cellular composition, activation states, and intercellular relationships within complex tissue microenvironments—information that is critical for accurate disease diagnosis and therapeutic decision-making.
Tyramide Signal Amplification (TSA) Technology
The principle of TSA is analogous to conventional DAB chromogenic IHC. TSA utilizes horseradish peroxidase (HRP)-conjugated secondary antibodies to catalyze the conversion of added fluorophore-conjugated tyramide substrates into highly reactive intermediates. These activated tyramide radicals form stable covalent bonds with tyrosine residues present on target antigens, resulting in the permanent deposition of fluorescent signals directly at the epitope site. Following signal development, heat-mediated epitope retrieval (HIER) is employed to strip non-covalently bound primary/secondary antibody complexes, thereby enabling sequential rounds of incubation with different primary antibodies and corresponding fluorophore-conjugated tyramide substrates. This iterative stripping-and-staining workflow facilitates robust multiplexed immunofluorescence labeling, allowing for the comprehensive spatial profiling of multiple biomarkers within a single tissue section.
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| Composition | TSA monochromatic fluorescent dye 540, 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). |
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| 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. |