储存缓冲液：DMF
Ready-to-use LumiDye™ 540 Tyramide (200×)是一款基于酪胺信号放大 (TSA)技术的即用型荧光检测试剂，酪胺信号放大技术利用辣根过氧化物酶 (HRP)催化酪胺底物在靶抗原附近沉积高密度标记物，从而实现对低丰度靶标的超灵敏原位检测。该技术突破了传统方法的检测极限，并显著降低了抗体用量。本产品的优势在于基于酪胺的信号放大技术能够提供极强的灵敏度、检测极微量的目的抗原。一方面，可与常规染色方法结合进行多色成像；另一方面，能够通过连续进行多个酪胺反应，对单一样本中的多种不同靶标进行多重标记。
酪胺信号放大技术是当前超灵敏原位检测领域的关键技术之一，凭借超灵敏检测和高分辨率保留的优势已广泛应用。本产品可适配免疫荧光、免疫组化、酶联免疫吸附实验及原位杂交等多种检测，既能与常规染色方法结合实现多色成像，又可通过连续酪胺反应对单一样本中的多种靶标进行多重标记，为复杂样本中低丰度蛋白或核酸的定位与分析提供有力工具。
注：效能等同于iFluor® 546 Tyramide (45103)。
注：本文中iFluor®为AAT Bioquest的商标及注册商标。
应用范围：多色荧光免疫组织化学原位杂交，酶联免疫吸附实验，神经科学稀有神经元标记，肿瘤生物学低丰度癌标志物检测等。
产品特点：
1、经济性：节省实验中所需的抗体用量95%以上；
2、高效省时：即用型设计，10 min内完成信号放大；
3、高灵敏性：可检测极低丰度靶标，灵敏度提升100倍；
4、多重标记：允许在同一样本中对多种不同靶标进行连续的酪胺反应和多重标记，为复杂样本的分析提供便利；
5、通用性：适用于免疫荧光、免疫组化、酶联免疫吸附实验及原位杂交等检测，无需调整核心操作流程。
产品参数：
1、Ex/Em：516/548 nm；
2、Molecular Weight：943.19；
产品组分：
| 组分 | 20 T | 200 T | | Ready-to-use LumiDye™ 540 Tyramide (200×) | 10 μL | 100 μL |
|
注：按1: 200稀释，每个样品使用100 μL染色液计算。
注意事项：
1、使用前，务必对产品进行瞬时离心，确保液体沉入管底后再开盖取用。
2、推荐Ready-to-use LumiDye™ 540 Tyramide起始稀释比为1: 200，浓度过高易致信号过强或背景升高，可以梯度测试 (从1: 100至1: 1000)寻找最佳浓度。
3、进行同一样本多靶标标记时，每一轮酪酰胺反应后，进行HRP淬灭或抗体剥离后再进行下一轮。
4、建议使用5 μg/mL的HRP结合物，降低浓度可能会显著减弱信号强度和灵敏度。
5、TSA试剂盒相比于荧光二抗灵敏度更高。因此，实验时使用较低的一抗可以降低非特异性结合带来的背景荧光，我们建议梯度设置一抗浓度以找浓度到最佳浓度。此外，我们建议设置阴性对照 (未与一抗孵育)以评估背景荧光的干扰，组织样本设置未染色对照 (不添加抗体或酪酰胺)，以确定组织自发荧光对背景的影响。
6、若做多色标记时，依据抗原密度选择染料 (低密度抗原用强信号染料；高密度抗原用较弱信号染料)。标记顺序可能影响结果，需自行摸索优化。
7、荧光染料易受光照影响而淬灭，请务必避光操作，以保持染料稳定性。
8、本产品仅限于科研用途并且不得存放于普通住宅内。
9、为了您的安全和健康，请遵循您所在常规实验室安全规定。
使用说明：
一、 实验前准备
1. 试剂准备：
(1) 将试剂从储存条件 (如4 °C)取出，平衡至室温 (15-25 °C)。
(2) 准备细胞培养基、PBS。
二、 操作步骤
1. 样本准备：
(1) 细胞样品
准备一份不孵育一抗的阴性对照样本 (可选)。首先，用PBS轻柔清洗细胞两次。随后，加入适量4%多聚甲醛 (pH7.4)溶液，于4 ℃固定细胞15 min，固定后再次用PBS清洗两次。接着，使用PBS配制的0.5% Triton X-100溶液在室温下通透细胞10 min，通透完成后，最后用PBS清洗细胞两次，即可进行后续操作。
(2) 石蜡组织切片
首先，将切片置于60 ℃的烘箱中30 min。随后，在通风橱中 (注：二甲苯有毒易挥发)，于室温下用二甲苯浸泡切片2次，每次5 min以彻底脱蜡。接着，室温下将切片浸没于无水乙醇中漂洗2次，每次5 min。之后，室温下依次浸没于梯度乙醇 (95%、90%、80%、70%)中各漂洗1次，每次5 min。漂洗后，室温下将切片浸入纯水漂洗3 min，再浸入PBS漂洗3 min，用滤纸小心吸干切片周围多余液体。用免疫组化笔沿组织边缘描绘轮廓以限定后续反应区域。进行抗原修复时，将0.1 M柠檬酸盐缓冲液 (pH 6.0)微波加热至沸腾，将处理好的切片完全浸没于缓冲液中，维持微沸状态10 min；修复完成后取出切片，室温下缓慢冷却 (注：不同样本可能需选择其他修复方法)。最后，用PBS清洗切片两次，即可进行后续操作。
2. (可选)内源性过氧化物酶灭活：
若需灭活，加入足量的3% Hydrogen Peroxide覆盖样品，室温孵育60 min。淬灭样品的内源性过氧化物酶活性。
3. (可选)内源性生物素阻断：
进行链霉亲和素/生物素检测时，我们建议阻断内源性生物素以降低背景。
注：使用标记LumiDye™ 染料的酪酰胺和HRP二抗的试剂盒可省略此步。
具体操作如下：首先，在室温下将样品与未标记的链霉亲和素溶液孵育15 min，随后用生物素封闭清洗缓冲液室温洗涤样品3次，每次5 min；接着，在室温下将样品与生物素溶液孵育30 min以封闭链霉亲和素上多余的生物素结合位点，孵育完毕后再用生物素封闭清洗缓冲液洗涤样品3次，每次5 min，即可进行后续实验。
4. 免疫标记：
(1) 首先，用封闭缓冲液室温封闭样品1 h。封闭后，用封闭缓冲液将一抗稀释至适当的浓度，将样品与一抗稀释液在室温孵育1 h或在4 ℃过夜 (注：使用含 HRP–Streptavidin 的试剂盒时，请选用生物素偶联的一抗)。随后，室温下用PBS洗涤样品3次，每次5 min。
(2) 洗涤后，在封闭缓冲液中将HRP-conjugated按1: 200稀释，用此稀释液室温孵育样品1 h。接着，再次室温下用PBS洗涤3次，每次5 min。之后，制备酪酰胺染色液：用1× Tyramide Amplification Buffer将200× Tyramide Stock Solution按1: 200稀释，每个样品需要100 μL染色液 (注：染色液在室温下避光保存不超过24 h)。将样品与染色液室温避光孵育10 min。
(3) 孵育完毕，室温下用PBS洗涤3次，每次5 min。最后，进行显微镜成像。组织切片需加盖玻片并封片后再成像。
三、 结果判读
定性分析（显微镜）：
图 1.大鼠外侧隔核背侧亚区、中间亚区和腹侧亚区免疫组织化学标记图
正常放大后荧光信号：细胞内可见反应区域有特异性荧光信号；
背景干扰：若细胞外或背景荧光强，提示洗涤不充分或染料浓度过高。
注：图片引自参考文献《Characterization of GABAergic neurons in the mouse lateral septum: a double fluorescence insituhybridization and immunohistochemical study using tyramide signal amplification》(doi: 10.1371/journal.pone.0073750)
常见疑问与解答：
1、问：Tyramide孵育时间能否延长？
答：不建议。标准孵育时间为室温避光10 min。延长孵育可能导致非特异性沉积，增大背景干扰。
2、问：信号微弱或无信号可能的原因？
答：HRP结合物浓度不足；一抗浓度过低或孵育时间不足 (需梯度优化)；抗原修复不充分；酪酰胺工作液失效 (避免反复冻融或过期)。
3、问：能否用于多重标记 (同一张切片多个靶标)？
答：可以，但需严格进行HRP淬灭：第一轮酪酰胺反应灭活残留HRP活性，再进行下一轮的孵育。
Ready-to-use Bodi Fluor 540 Tyramide (200×) is a ready-to-use fluorescent detection reagent based on Tyramide Signal Amplification (TSA) technology. TSA technology utilizes horseradish peroxidase (HRP) to catalyze the deposition of high-density labels from tyramide substrates in the vicinity of target antigens, enabling ultrasensitive in situ detection of low-abundance targets. This technology breaks through the detection limits of conventional methods and significantly reduces antibody consumption. The advantage of this product lies in the tyramide-based signal amplification technology, which provides exceptional sensitivity for detecting trace amounts of target antigens. On one hand, it can be combined with conventional staining methods for multicolor imaging; on the other hand, multiple different targets in a single sample can be multiplex-labeled by performing consecutive tyramide reactions.
Tyramide signal amplification is one of the key technologies in the field of ultrasensitive in situ detection, and has been widely applied due to its advantages of ultrasensitive detection and high-resolution preservation. This product is compatible with various assays including immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization. It can be combined with conventional staining methods to achieve multicolor imaging, and can also perform multiplex labeling of multiple targets in a single sample through consecutive tyramide reactions, providing a powerful tool for the localization and analysis of low-abundance proteins or nucleic acids in complex samples.
Note: Performance is equivalent to iFluor® 546 Tyramide (45103).
Note: iFluor® is a trademark and registered trademark of AAT Bioquest.
Application Scope
Multicolor fluorescent immunohistochemistry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), labeling of rare neurons in neuroscience, detection of low-abundance cancer biomarkers in tumor biology, etc.
Product Features
1. Cost-effectiveness: Saves more than 95% of the antibody required in experiments;
2. Efficiency and time-saving: Ready-to-use design, completing signal amplification within 10 minutes;
3. High sensitivity: Capable of detecting extremely low-abundance targets, with a 100-fold increase in sensitivity;
4. Multiplex labeling: Allows consecutive tyramide reactions and multiplex labeling of multiple different targets in the same sample, facilitating the analysis of complex samples;
5. Versatility: Suitable for immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization without the need to adjust core operational procedures.
Product Parameters
1. Ex/Em: 516/548 nm;
2. Molecular Weight: 943.19;
Product Components
| Component | 20 T | 200 T | Ready-to-use LumiDye™ 540 Tyramide (200×)
| 10 μL | 100 μL |
|
Note: Calculated based on a 1:200 dilution, with 100 μL of staining solution used per sample.
Precautions
1. Before use, centrifuge the product briefly to ensure the liquid settles at the bottom of the tube before opening the cap for use.
2. The recommended starting dilution ratio for Ready-to-use Bodi Fluor 540 Tyramide is 1:200. Excessively high concentrations may lead to overly strong signals or elevated background. Gradient testing (from 1:100 to 1:1000) is recommended to find the optimal concentration.
3. When performing multi-target labeling on the same sample, after each round of tyramide reaction, perform HRP quenching or antibody stripping before proceeding to the next round.
4. It is recommended to use 5 μg/mL HRP conjugates; lower concentrations may significantly reduce signal intensity and sensitivity.
5. TSA kits exhibit higher sensitivity compared to fluorescent secondary antibodies. Therefore, using a lower primary antibody concentration in experiments can reduce background fluorescence caused by non-specific binding. We recommend setting a primary antibody concentration gradient to find the optimal concentration. In addition, we recommend setting a negative control (not incubated with primary antibody) to assess the interference of background fluorescence, and an unstained control (without adding antibody or tyramide) for tissue samples to determine the effect of tissue autofluorescence on the background.
6. When performing multicolor labeling, select dyes based on antigen density (use strong-signal dyes for low-density antigens; use weak-signal dyes for high-density antigens). The labeling order may affect results and requires optimization through preliminary experiments.
7. Fluorescent dyes are susceptible to photobleaching due to light exposure. Be sure to perform operations in the dark to maintain dye stability.
8. This product is for research use only and must not be stored in ordinary residential premises.
9. For your safety and health, please follow the standard laboratory safety regulations in your institution.
Instructions for Use
I. Pre-Experiment Preparation
1. Reagent Preparation:
(1) Remove reagents from storage conditions (e.g., 4 °C) and equilibrate to room temperature (15-25 °C).
(2) Prepare cell culture medium and PBS.
II. Operational Steps
1. Sample Preparation:
(1) Cell Samples
Prepare a negative control sample that is not incubated with primary antibody (optional). First, gently wash the cells twice with PBS. Then, add an appropriate amount of 4% paraformaldehyde (pH 7.4) solution and fix the cells at 4 °C for 15 minutes. After fixation, wash the cells twice again with PBS. Next, permeabilize the cells with 0.5% Triton X-100 solution prepared in PBS at room temperature for 10 minutes. After permeabilization, wash the cells twice with PBS to proceed to subsequent operations.
(2) Paraffin Tissue Sections
First, place the sections in an oven at 60 °C for 30 minutes. Then, in a fume hood (Note: xylene is toxic and volatile), immerse the sections in xylene at room temperature twice for 5 minutes each to completely dewax. Next, immerse the sections in absolute ethanol at room temperature twice for 5 minutes each. After that, immerse the sections in graded ethanol (95%, 90%, 80%, 70%) at room temperature once for 5 minutes each. After rinsing, immerse the sections in pure water at room temperature for 3 minutes, then in PBS for 3 minutes, and carefully absorb excess liquid around the sections with filter paper. Use an immunohistochemistry pen to draw a contour along the edge of the tissue to define the subsequent reaction area. For antigen retrieval, microwave 0.1 M citrate buffer (pH 6.0) until boiling, completely immerse the processed sections in the buffer, and maintain a slight boil for 10 minutes; after retrieval, remove the sections and allow them to cool slowly at room temperature (Note: different samples may require alternative retrieval methods). Finally, wash the sections twice with PBS to proceed to subsequent operations.
2. (Optional) Endogenous Peroxidase Inactivation:
If inactivation is required, add an adequate amount of 3% Hydrogen Peroxide to cover the sample and incubate at room temperature for 60 minutes to quench the endogenous peroxidase activity of the sample.
3. (Optional) Endogenous Biotin Blocking:
When performing streptavidin/biotin detection, we recommend blocking endogenous biotin to reduce background.
Note: This step can be omitted when using kits with tyramide labeled with Bodi Fluor dyes and HRP secondary antibodies.
Specific operations are as follows: First, incubate the sample with unlabeled streptavidin solution at room temperature for 15 minutes, then wash the sample with biotin blocking wash buffer at room temperature 3 times for 5 minutes each; next, incubate the sample with biotin solution at room temperature for 30 minutes to block excess biotin binding sites on streptavidin. After incubation, wash the sample with biotin blocking wash buffer 3 times for 5 minutes each to proceed to subsequent experiments.
4. Immunolabeling:
(1) First, block the sample with blocking buffer at room temperature for 1 hour. After blocking, dilute the primary antibody to an appropriate concentration with blocking buffer, and incubate the sample with the primary antibody dilution at room temperature for 1 hour or at 4 °C overnight (Note: When using kits containing HRP-Streptavidin, please use biotin-conjugated primary antibodies). Then, wash the sample with PBS at room temperature 3 times for 5 minutes each.
(2) After washing, dilute HRP-conjugated secondary antibody 1:200 in blocking buffer, and incubate the sample with this dilution at room temperature for 1 hour. Then, wash the sample with PBS at room temperature 3 times for 5 minutes each. Next, prepare tyramide staining solution: dilute the 200× Tyramide Stock Solution 1:200 with 1× Tyramide Amplification Buffer, requiring 100 μL of staining solution per sample (Note: The staining solution should not be stored at room temperature in the dark for more than 24 hours). Incubate the sample with the staining solution at room temperature in the dark for 10 minutes.
(3) After incubation, wash the sample with PBS at room temperature 3 times for 5 minutes each. Finally, perform microscopic imaging. Tissue sections need to be coverslipped and mounted before imaging.
III. Result Interpretation
Qualitative Analysis (Microscopy):
Figure 1. Immunohistochemical labeling of the dorsal, intermediate, and ventral subregions of the rat lateral septal nucleus
Fluorescent signal after normal magnification: Specific fluorescent signals are visible in the reactive regions within the cells;
Background interference: Strong extracellular or background fluorescence indicates insufficient washing or excessively high dye concentration.
Note: The image is reproduced from the reference "Characterization of GABAergic neurons in the mouse lateral septum: a double fluorescence in situ hybridization and immunohistochemical study using tyramide signal amplification" (doi: 10.1371/journal.pone.0073750).
Frequently Asked Questions and Answers
1. Q: Can the tyramide incubation time be extended?
A: It is not recommended. The standard incubation time is 10 minutes at room temperature in the dark. Prolonged incubation may lead to non-specific deposition and increased background interference.
2. Q: What are the possible causes of weak or no signal?
A: Insufficient HRP conjugate concentration; primary antibody concentration too low or incubation time insufficient (gradient optimization required); insufficient antigen retrieval; tyramide working solution expired (avoid repeated freeze-thaw cycles or use after expiration).
3. Q: Can it be used for multiplex labeling (multiple targets on the same section)?
A: Yes, but HRP quenching must be strictly performed: inactivate residual HRP activity after the first round of tyramide reaction before proceeding to the next round of incubation.
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