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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