Four Color Multiplex Fluorescent Immunostaining Kit (Mouse Rabbit)

The tissue microenvironment contains complex cell components that hold great biological and clinical significance in terms of phenotype, state, abundance, and distribution. To visualize these cells within the tissue, immunohistochemical staining with antibody markers is commonly used. However, traditional IHC detection can only display a single characteristic, limiting our ability to understand the complexity of cell composition, states, and relationships in the microenvironment. This information is crucial for disease diagnosis and treatment.
To address this issue, tyrosine signal amplification (TSA) technology has been developed. Similar to the conventional DAB coloring method used in immunohistochemistry, TSA utilizes HRP-labeled secondary antibodies. HRP catalyzes the addition of fluorescent substrates to the system, generating activated fluorescent substrates that can bind covalently to antigens. This covalent binding ensures that the sample is permanently marked with fluorescence. Subsequently, the non-covalently bound antibodies are washed away using heat repair, allowing for the second round of incubation with another primary antibody and fluorescent substrate. This cycle can be repeated, enabling the labeling of multiple characteristics.
By harnessing the power of TSA technology, we can gain valuable insights into the composition, state, and relationships of cells in the intricate tissue microenvironment. This information not only enhances our understanding of biological processes but also holds immense potential for advancing disease diagnosis and treatment.

This method is primarily employed to stain human or mouse tissues, paraffin sections, and TMA chips for immunohistochemical analysis.

To create a dye solution, there are different methods depending on the form of the dye. If the dye is in dry powder form, you can dissolve it by adding 100ul of DMSO. This will result in a 100X concentrated dye mother liquor. On the other hand, if the dye is already in liquid form, it means that it was originally dissolved in DMSO at the factory to create a 100X mother liquor.
For the dye working solution, it needs to be appropriately diluted for current use. In the case of the signal amplification reaction solution, you would dilute it in a ratio of 1:100. This means that you would mix 1 part of the concentrated dye mother liquor with 100 parts of the signal amplification reaction solution to prepare the working solution.
Similarly, for DAPI, which is another type of dye, it should be diluted using sterile water. To create the working solution, you would dilute it in a ratio of 1:100. This involves mixing 1 part of the concentrated DAPI with 100 parts of sterile water.
In summary, the process of preparing dye solutions involves different dilution methods depending on the form of the dye. It is important to follow the correct ratios and use appropriate solvents to ensure the desired concentration is achieved for the working solutions.
When using the kit, it's important to note that the standard configuration includes a pika universal secondary antibody that is suitable for human samples, but not for mouse tissue. Therefore, before conducting the experiment, it's crucial to verify that the primary antibody species matches properly. In the case of mouse tissues, it's necessary to replace the secondary antibodies with anti-rabbit ones to ensure accuracy and reliable results. It's essential to follow these guidelines to ensure that the experiment is successful and valid.

The sole purpose of using this kit is for immunohistochemistry procedures and it should not be employed for any other application. Please make sure to adhere to this guideline for optimal results.
2. This kit is for professional use only.
3. Appropriate protective measures should be adopted to avoid contact of reagents with skin and eyes.
4. The activity of reagents beyond the expiration date may be reduced, so reagents beyond the expiration date should not be used.
5. If the dyeing components of this kit are mixed with products of other companies, abnormalities may occur during the dyeing process.
6. Incomplete dewaxing can easily affect the dyeing effect.
7. In order to prevent possible false negative and false positive results, a positive control and a negative control must be performed simultaneously during the experiment.
8. All kinds of waste generated in the use of this kit should be treated in accordance with the "Medical Waste Management Regulations".

Fluorescent dyes should be stored in the dark at 2～8° C. The validity period is 12 months from receipt.

Soluble in water and ethanol.

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4. Qian Bangguo. Jiao Lei. The application of multi-label immunofluorescence staining and Doppler imaging technology in histological research. Chinese Journal of Histochemistry and Cytochemistry, 2017 (4): 373-382.