Six Color Multiplex Fluorescent Immunostaining Kit (Mouse Rabbit)

The tissue microenvironment contains intricate components of cell structures that play a vital role in biological processes and have significant clinical implications. To visualize and understand these cells, their phenotype, state, abundance, and distribution, antibody staining is commonly employed through immunohistochemical (IHC) techniques. However, traditional IHC detection falls short in displaying multiple indices, making it challenging to comprehend the complex composition, states, and interrelationships of cells in the tissue microenvironment. This information holds paramount importance in disease diagnosis and treatment.
To address this limitation, a principle called tyrosine signal amplification (TSA) technology has been developed. It follows a similar approach to the conventional DAB coloring method used in immunohistochemistry, utilizing HRP-labeled secondary antibodies. The HRP enzyme catalyzes the addition of fluorescent substrates, leading to the generation of activated fluorescent compounds. These compounds covalently bind to the antigens present in the sample, enabling the sample to be covalently linked with fluorescence tags. Subsequently, a heat repair method is employed to eliminate non-covalently bound antibodies, followed by a second round of incubation with another primary antibody and fluorescent substrate. This cyclic process allows for multiple labeling and visualization of various targets within the tissue, effectively unraveling the complexity of the tissue microenvironment.

To dilute fluorescent dye, a dry powder form can be dissolved in 100ul of DMSO to create a 100X dye mother liquor. If the dye is already in liquid form, it was likely prepared as a 100X mother liquor dissolved in DMSO at the factory. The working solution for the dye can be prepared by diluting 1:100 using the signal amplification reaction solution, ensuring it is ready for current use. DAPI, on the other hand, requires sterile water to be used for dilution purposes. In this case, a working solution can be prepared by diluting 1:100. These preparation methods ensure accurate and consistent results when using fluorescent dye in experimental procedures.
To ensure accurate results, it is important to use the appropriate secondary antibody for your samples. The kit comes with a pika universal secondary antibody that is ideal for human tissue, but not suitable for mouse tissue. Before beginning your experiment, it is crucial to verify that the primary antibody species matches with the secondary antibody provided in the kit. If you will be using mouse tissue, you will need to replace the standard secondary antibody with a different type that is compatible with your samples. This will ensure that your experiment yields the most accurate and reliable data possible.

The recommended storage condition for FluorFluorescent dyes is at 2～8° C in a dark environment. It is important to note that the validity period is 12 months from the date of receipt.

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