酪酰胺信号放大 (TSA，Tyramide signal amplification)技术是一类利用辣根过氧化酶（HRP）对靶蛋白进行标记的酶学检测方法，类似常规免疫组化的DAB显色方法，TSA技术同样采用HRP标记的二抗，同样有对应的“显色”步骤（HRP催化加入反应体系的酪胺荧光素底物，产生活化荧光底物，活化底物可与抗原上的酪氨酸等残基共价结合，使样品上稳定的共价结合酪胺荧光素。之后用热修复法或者抗体洗脱液洗去非共价结合的一抗-二抗-HRP复合物，重复下一种一抗-HRP二抗来第二轮孵育，换另一种酪胺荧光素底物，如此往复就可实现多重标记。
TSA详细原理是利用酪胺Tyramide的过氧化物酶反应 (酪胺盐在HRP催化H₂O₂下形成共价键结合位点)，产生大量的酶促产物，该产物能与周围的蛋白残基(包括色氨酸、组氨酸和酪氨酸残基)结合，这样在抗原-抗体结合部位就有大量的荧光素沉积，结果使其检测信号得到10-100倍增强。简单来说，用这种方法做多重免疫荧光是利用二抗上带有的HRP（而不是直接偶联荧光素），来催化后续添加入体系的非活性荧光素。荧光素在HRP和过氧化氢的作用下被活化，跟临近蛋白的酪氨酸残基共价偶联，使得蛋白样品与荧光素稳定结合。然后微波或煮沸或者水浴等热修复处理，前一轮非共价结合的抗体被洗掉，共价结合的荧光素稳定共价结合在样本切片蛋白上。再换个一抗来第二轮孵育，周而复始。等到所有抗体孵育结束，荧光素都结合好后，最后去检测结果。由于每次体系中都只有单一抗体孵育，因此无需担心抗体交叉反应，以及一抗二抗种属匹配问题，大大减少了实验设计时不同种属抗体选择匹配的限制。也就是说，如果用TSA技术，同一张片子上所有的靶标都可以选用特异性高的兔单克隆抗体。搭配同一支抗兔的HRP二抗就可以进行实验，而且信号放大的倍数大大增强。本公司开发的试剂盒具体酪胺荧光染料为以下其中一种或者多种：TYR-480，TYR-520，TYR-570，TYR-620，TYR-700，TYR-780、TYR-650、TYR-540。此试剂盒中的荧光染料可单独或配合使用。可以实现单标、双标、三标以及更多重荧光放大/多重同源抗体荧光标记等功能，极大丰富了此试剂盒的内涵。
试剂盒组成：
E1372805
| 组分名称
| 20T
| Storage
|
E1372805A
| TYR-480 荧光染料（浓缩型，200×）
| 10μL
| 2-8 ℃
|
E1372805B
| TYR-520 荧光染料（浓缩型，200×）
| 10μL
| 2-8 ℃
|
E1372805C
| TYR-570 荧光染料（浓缩型，200×）
| 10μL
| 2-8 ℃
|
E1372805D
| TYR-620 荧光染料（浓缩型，200×）
| 10μL
| 2-8 ℃
|
E1372805E
| TYR-700 荧光染料（浓缩型，200×）
| 10μL
| 2-8 ℃
|
E1372805F
| TYR-780 荧光染料（浓缩型，200×）
| 10μL
| 2-8 ℃
|
E1372805G
| TYR-650 荧光染料（浓缩型，200×）
| 10μL
| 2-8 ℃
|
E1372805H
| TSA buffer
| 11.2mL
| 2-8 ℃
|
E1372805I
| HRP 山羊抗兔二抗
| 11.2mL
| 2-8 ℃
|
E1372805J
| Dapi 染液（即用型）
| 2mL
| 2-8 ℃
|
E1372805K
| 抗体稀释液
| 14mL
| 2-8 ℃
|
E1372805L
| 3%过氧化氢
| 14mL
| 2-8 ℃
|
E1372805M
| 抗荧光淬灭封片剂
| 2mL
| 2-8 ℃ |
注：产品包装规格以TSA Buffer为基准，1T对应TSA Buffer的定量为80μL。
*染料使用方法：TSA荧光染料反应液=TYR-xxx 荧光染料+TSA buffer；TYR-xxx 荧光染料：TSA buffer=1：200；TYR-xxx 荧光染料与TSA buffer的稀释比例可以根据具体情况灵活调整优化，最佳范围为1:50--1:400(1:200大部分情况能得到最佳结果）；一般建议若一抗孵育时间常温1h-3h内，建议稀释比例1:50-1:200,若一抗孵育时间4℃过夜（12h或以上），建议稀释比例1:200-1:400或更高。
染料
| 激发波长
| 发射波长
|
DAPI 蓝色
| 350 nm
| 420 nm
|
TYR-480 荧光染料（浓缩型，200×）
| 450 nm
| 480 nm
|
TYR-520 荧光染料（浓缩型，200×）
| 490 nm
| 520 nm
|
TYR-570 荧光染料（浓缩型，200×）
| 550 nm
| 570 nm
|
TYR-620 荧光染料（浓缩型，200×）
| 590 nm
| 620 nm
|
TYR-690 荧光染料（浓缩型，200×）
| 640 nm
| 690 nm
|
TYR-780 荧光染料（浓缩型，200×）
| 750 nm
| 780 nm
|
TYR-700 荧光染料（浓缩型，200×）
| 680 nm
| 700 nm
|
TYR-650 荧光染料（浓缩型，200×）
| 630 nm
| 650 nm
|
TYR-540 荧光染料（浓缩型，200×）
| 515 nm
| 540 nm |
操作流程简图
详细操作步骤
1、样本准备：
1）石蜡切片：依次将切片放入二甲苯Ⅰ 15min-二甲苯Ⅱ 15min-无水乙醇Ⅰ 5min-无水乙醇Ⅱ 5min-95%乙醇 5min-85%乙醇 5min-75%乙醇5min，蒸馏水洗。
2）冰冻切片：冰冻切片固定10-30min，PBS洗5min，重复3次，滴加0.3% triton-X100破膜液通透20min，PBS洗5min，重复3次。
3）细胞爬片或者细胞涂片：细胞样本固定10-30min，PBS洗5min重复3次，滴加0.3% triton-X100破膜液通透20min，PBS洗5min，重复3次。
2、抗原修复：
组织切片置于盛满PH9.0 EDTA碱性抗原修复液或者PH6.0柠檬酸修复缓冲液的修复盒中于微波炉内进行抗原修复（也可以用高压1-2min 100℃水煮15min，95℃水浴20min等其他热修复方法）。中火8min，停火8min，转中低火7min，此过程中应防止缓冲液过度蒸发，切勿干片。自然冷却后将玻片置于PBS (pH7.4) 中在脱色摇床上晃动洗涤3次，每次5min。(修复液和修复条件根据组织类型以及抗原类型来确定，冰冻切片和细胞样本可省略此步骤。抗原修复液推荐使用EDTA 9.0抗原修复液和柠檬酸抗原修复液。
3、阻断内源性过氧化物酶：切片放入3%过氧化氢溶液，室温避光孵育15min，将玻片置于PBS (pH7.4) 中在脱色摇床上晃动洗涤3次，每次5min。
4、非特异性靶点封闭:切片稍甩干后用组化笔在组织周围画圈（防止抗体流走），在圈内滴加用抗体稀释液（或者其他封闭液3%BSA或者山羊血清）均匀覆盖组织，室温封闭30min。额外说明：抗体稀释液内含有各种保护剂以及防腐剂，可以用来封闭或者稀释一抗，稀释后的一抗可以长期4℃保存（在常温下也可以保存一个月之久）
5、加一抗：轻轻甩掉封闭液，在切片上滴加用抗体稀释液稀释好的的一抗X，切片平放于避光湿盒内4°C孵育过夜或者37°C 1-2h（湿盒内加少量水防止抗体蒸发）；
6、加HRP二抗：玻片置于PBS (pH7.4) 中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后在圈内滴加与一抗相应种属的HRP二抗覆盖组织，避光室温孵育50min，PBS洗三次。额外说明：本试剂盒内自带的HRP山羊抗兔二抗为即用型，具有超高灵敏度，随时可用，无需配置。
7、TSA荧光染料反应液反应：浓缩型荧光染料与TSA buffer按照1:50-1:200的比例混合均匀，切片滴加配好的TSA荧光染料反应液均匀覆盖组织室温反应1-15min（最佳时间5min-10min），PBS洗三次（预实验可先染1min洗干净荧光染料后显微镜下观察染色效果，如果阳性弱继续滴加荧光染料加强染色强度直至合适强度后继续进行下一步）。
8、抗体洗脱：石蜡切片置于抗原修复液中95℃水浴25-40min（根据不同抗体亲和力灵活调整时间）或者滴加适量37℃预热至完全溶解的mIHC专用抗体洗脱液（冰冻切片爬片骨组织建议用）覆盖样本，37℃放置5-20min，弃去洗脱液，再次滴加适量抗体洗脱液覆盖样本37℃放置5-20min，弃洗脱液，PBS洗三次，每次5min（石蜡切片可用热修复洗脱或者抗体洗脱液洗脱，细胞及冰切切片需用抗体洗脱液进行洗脱）
9、重复3-8步骤（换用另外一种TYR-XXX 荧光染料）---第二轮标记
10、重复3-8步骤（换用另外一种TYR-XXX 荧光染料）---第三轮标记
11、重复3-8步骤（换用另外一种TYR-XXX 荧光染料）---第四轮标记
12、重复3-8步骤（换用另外一种TYR-XXX 荧光染料）---第五轮标记
13、重复3-8步骤（换用另外一种TYR-XXX 荧光染料）---第六轮标记
14、重复3-7步骤（换用另外一种TYR-XXX 荧光染料）---第七轮标记
注意：如果最后一轮染780染料，可以使用EDTA或柠檬酸进一步修复，以降低背景。
15、DAPI复染细胞核：玻片置于PBS (pH7.4) 中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后在圈内滴加DAPI即用型染液，避光室温孵育5min-20min。
16、封片：玻片置于PBS (pH7.4) 中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后用抗荧光淬灭封片剂封片。
17、切片于荧光显微镜/共聚焦/多通道荧光扫描仪/多光谱成像系统下观察并采集图像。
Tyramide signal amplification (TSA) is an enzymatic detection method for labeling target proteins using horseradish peroxidase (HRP). Similar to the conventional DAB chromogenic method for immunohistochemistry, TSA also employs HRP-conjugated secondary antibodies and includes a corresponding “development” step. HRP catalyzes the tyramide-fluorophore substrate added into the reaction system to generate activated fluorophore substrates, which can covalently bind to tyrosine and other residues on antigens, resulting in stable covalent attachment of fluorophore tyramide to the sample. Subsequently, the non-covalently bound primary antibody–secondary antibody–HRP complex is removed via heat-induced antigen retrieval or antibody stripping buffer. The sample is then incubated with another primary antibody and HRP-conjugated secondary antibody for the second round, followed by a different tyramide-fluorophore substrate. Repeating this process enables multiplex labeling.
The detailed principle of TSA relies on the peroxidase reaction of tyramide: tyramide salts form covalent binding sites under HRP catalysis in the presence of H₂O₂, generating abundant enzymatic products. These products bind to surrounding protein residues (including tryptophan, histidine and tyrosine residues). Consequently, massive fluorophores are deposited at the antigen-antibody binding sites, amplifying the detection signal by 10–100 fold. Simply put, multiplex immunofluorescence with this method uses HRP conjugated to secondary antibodies (instead of directly conjugated fluorophores) to catalyze inactive fluorophores added later to the reaction mix. The fluorophores are activated by HRP and hydrogen peroxide and covalently couple to tyrosine residues on adjacent proteins, forming stable linkage between fluorophores and proteins in the specimen. Next, heat treatment such as microwave heating, boiling or water bath incubation strips off the non-covalently bound antibodies from the previous round, while the covalently attached fluorophores remain stably anchored to proteins on the tissue section. The sample is then incubated with a new primary antibody for the next cycle, and the procedure is repeated. Once all antibody incubations are completed and fluorophores are immobilized, the signals are detected. Since only one primary antibody is incubated in each round, there is no concern about antibody cross-reactivity or primary/secondary antibody species matching issues. This greatly reduces constraints on species selection during experimental design. In other words, with TSA technology, highly specific rabbit monoclonal antibodies can be used for all targets on the same section. The experiment can be performed with the same HRP-conjugated anti-rabbit secondary antibody, and the signal is substantially amplified. The tyramide fluorophores provided in our kit include one or more of the following: TYR-480, TYR-520, TYR-570, TYR-620, TYR-700, TYR-780, TYR-650, TYR-540. These fluorophores can be used individually or in combination. The kit supports single-plex, duplex, triplex and higher-order multiplex fluorescence amplification, as well as multiplex labeling with primary antibodies from the same host species, which greatly expands the application scope of this kit.
Contents & Storage:
| Component No. | Component Name | Appearance | 20T | Storage |
| E1372805A | TYR-480 Fluorescent Dye (Concentrated, 200×) | Liquid | 10μL | 2-8 ℃ |
| E1372805B | TYR-520 Fluorescent Dye (Concentrated, 200×) | Liquid | 10μL | 2-8 ℃ |
| E1372805C | TYR-570 Fluorescent Dye (Concentrated, 200×) | Liquid | 10μL | 2-8 ℃ |
| E1372805D | TYR-620 Fluorescent Dye (Concentrated, 200×) | Liquid | 10μL | 2-8 ℃ |
| E1372805E | TYR-700 Fluorescent Dye (Concentrated, 200×) | Liquid | 10μL | 2-8 ℃ |
| E1372805F | TYR-780 Fluorescent Dye (Concentrated, 200×) | Liquid | 10μL | 2-8 ℃ |
| E1372805G | TYR-650 Fluorescent Dye (Concentrated, 200×) | Liquid | 10μL | 2-8 ℃ |
| E1372805H | TSA buffer | Liquid | 11.2mL | 2-8 ℃ |
| E1372805I | HRP Goat Anti-Rabbit Secondary Antibody
| Liquid | 11.2mL | 2-8 ℃ |
| E1372805J | DAPI Staining Solution (Ready-to-Use)
| Liquid | 2mL | 2-8 ℃ |
| E1372805K | Antibody Diluent
| Liquid | 14mL | 2-8 ℃ |
| E1372805L | 3% H₂O₂ | Liquid | 14mL | 2-8 ℃ |
| E1372805M | Anti-Fade Mounting Medium
| Liquid | 2mL | 2-8 ℃ |
Note: The kit packaging specification is based on TSA Buffer. 1 T corresponds to a defined volume of 80 μL TSA Buffer.
Dye preparation protocol:
TSA fluorescent dye working solution = TYR‑xxx fluorophore + TSA Buffer; the volume ratio of TYR‑xxx fluorophore to TSA Buffer is 1:200. The dilution ratio of TYR‑xxx fluorophore in TSA Buffer can be flexibly adjusted and optimized according to experimental conditions. The optimal range is 1:50–1:400 (1:200 yields optimal results in most cases). In general, if the primary antibody is incubated at room temperature for 1–3 h, a dilution ratio of 1:50–1:200 is recommended. If the primary antibody is incubated overnight at 4 °C (12 h or longer), a dilution ratio of 1:200–1:400 or higher is recommended.
Dye
| Excitation Wavelength
| Emission Wavelength
|
| DAPI Blue | 350 nm | 420 nm |
| TYR-480 Fluorophore (Concentrated, 200×) | 450 nm | 480 nm |
| TYR-520 Fluorophore (Concentrated, 200×) | 490 nm | 520 nm |
| TYR-570 Fluorophore (Concentrated, 200×) | 550 nm | 570 nm |
| TYR-620 Fluorophore (Concentrated, 200×) | 590 nm | 620 nm |
| TYR-690 Fluorophore (Concentrated, 200×) | 640 nm | 690 nm |
| TYR-780 Fluorophore (Concentrated, 200×) | 750 nm | 780 nm |
| TYR-700 Fluorophore (Concentrated, 200×) | 680 nm | 700 nm |
| TYR-650 Fluorophore (Concentrated, 200×) | 630 nm | 650 nm |
| TYR-540 Fluorophore (Concentrated, 200×) | 515 nm | 540 nm |
Schematic Operating Procedure:
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Detailed Operating Procedures:
1. Sample Preparation
① Paraffin sections: Sequentially immerse the sections in Xylene I for 15 min → Xylene II for 15 min → Absolute Ethanol I for 5 min → Absolute Ethanol II for 5 min → 95% Ethanol for 5 min → 85% Ethanol for 5 min → 75% Ethanol for 5 min, then rinse with distilled water.
② Frozen sections: Fix frozen sections for 10–30 min, wash with PBS for 5 min, repeat 3 times. Add 0.3% Triton X-100 permeabilization solution and incubate for 20 min, wash with PBS for 5 min, repeat 3 times.
③ Cell coverslips or cell smears: Fix cell samples for 10–30 min, wash with PBS for 5 min, repeat 3 times. Add 0.3% Triton X-100 permeabilization solution and incubate for 20 min, wash with PBS for 5 min, repeat 3 times.
2. Antigen Retrieval
Place tissue sections in a retrieval box filled with EDTA alkaline antigen retrieval buffer (pH 9.0) or citrate antigen retrieval buffer (pH 6.0), and perform antigen retrieval in a microwave oven. (Alternative thermal retrieval methods are also acceptable: high-pressure treatment for 1–2 min, boiling at 100 °C for 15 min, or water bath at 95 °C for 20 min.)
Microwave setting: medium heat for 8 min, stop heating for 8 min, then medium-low heat for 7 min. Prevent excessive evaporation of the buffer and avoid drying out the sections. After natural cooling, place the slides in PBS (pH 7.4) and wash on a decolorizing shaker 3 times, 5 min each time.
(The retrieval buffer and retrieval conditions should be selected according to tissue type and target antigen. This step can be omitted for frozen sections and cell samples. EDTA retrieval buffer (pH 9.0) and citrate retrieval buffer are recommended.)
3. Blocking of Endogenous Peroxidase
Immerse sections in 3% H₂O₂ solution and incubate at room temperature in the dark for 15 min. Wash slides in PBS (pH 7.4) on a decolorizing shaker 3 times, 5 min each time.
4. Non‑specific Target Blocking
Gently shake off excess liquid from sections. Use a immunohistochemistry pen to draw a circle around the tissue to prevent antibody outflow. Add Antibody Diluent (or alternative blocking reagents such as 3% BSA or goat serum) inside the circle to fully cover the tissue, and block at room temperature for 30 min.
Additional note: The Antibody Diluent contains protective agents and preservatives. It can be used for blocking or primary antibody dilution. Diluted primary antibody can be stored long‑term at 4 °C, and is stable for up to one month at room temperature.
5. Primary Antibody Incubation
Remove blocking buffer. Add primary antibody X diluted with antibody dilution buffer onto the section. Place the section flat in a light‑tight humidified chamber and incubate at 4 °C overnight, or at 37 °C for 1–2 h (add a small amount of water in the humidified chamber to prevent antibody evaporation).
6. HRP‑Conjugated Secondary Antibody Incubation
Wash slides in PBS (pH 7.4) on a decolorizing shaker 3 times, 5 min each time. Gently shake off excess liquid, add HRP Goat Anti-Rabbit Secondary Antibody matched to the primary antibody species to cover the tissue, incubate at room temperature in the dark for 50 min, then wash with PBS three times.
Additional note: The HRP goat anti‑rabbit secondary antibody supplied in this kit is ready‑to‑use with ultra‑high sensitivity; no preparation is required.
7. TSA Fluorophore Working Solution Reaction
Mix the concentrated fluorophore with TSA Buffer at a ratio of 1:50–1:200. Add the prepared TSA fluorophore working solution onto the section to fully cover the tissue, incubate at room temperature for 1–15 min (optimal incubation time: 5–10 min), then wash with PBS three times.
(Preliminary test suggestion: After 1 min of staining, wash off the fluorophore and check the staining signal under a microscope. If the positive signal is weak, add more fluorophore working solution to enhance signal intensity until satisfactory, then proceed to the next step.)
8. Antibody Stripping
For paraffin sections: Immerse in antigen retrieval buffer and incubate in a 95 °C water bath for 25–40 min (adjust time flexibly based on antibody affinity). Alternatively, apply pre‑warmed mIHC‑specific antibody stripping buffer (fully dissolved at 37 °C) to cover the sample (recommended for frozen sections, cell coverslips and bone tissues), incubate at 37 °C for 5–20 min, discard the stripping buffer, reapply fresh stripping buffer and incubate at 37 °C for another 5–20 min. Discard buffer and wash with PBS three times, 5 min each time.
(For paraffin sections, either thermal retrieval stripping or antibody stripping buffer can be used. Antibody stripping buffer is mandatory for cell samples and frozen sections.)
9. Repeat Steps 3–8 (use a different TYR‑XXX fluorophore) — Round 2 Labeling
10. Repeat Steps 3–8 (use a different TYR‑XXX fluorophore) — Round 3 Labeling
11. Repeat Steps 3–8 (use a different TYR‑XXX fluorophore) — Round 4 Labeling
12. Repeat Steps 3–8 (use a different TYR‑XXX fluorophore) — Round 5 Labeling
13. Repeat Steps 3–8 (use a different TYR‑XXX fluorophore) — Round 6 Labeling
14. Repeat Steps 3–7 (use a different TYR‑XXX fluorophore) — Round 7 Labeling
Note: If the last round uses TYR‑780 fluorophore, further retrieval with EDTA or citrate buffer can be performed to reduce background.
15. DAPI Counterstaining of Nuclei
Wash slides in PBS (pH 7.4) on a decolorizing shaker 3 times, 5 min each time. Gently shake off excess liquid, add ready‑to‑use DAPI Staining Solution inside the circle, incubate at room temperature in the dark for 5–20 min.
16. Mounting
Wash slides in PBS (pH 7.4) on a decolorizing shaker 3 times, 5 min each time. Gently shake off excess liquid, and mount sections with Anti-Fade Mounting Medium.
17. Image Acquisition
Observe and capture images under a fluorescence microscope / confocal microscope / multi‑channel fluorescence scanner / multispectral imaging system.
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