mIHC Complete TSA Multiplex Kit - 5 Color (A334670)

mIHC Complete TSA Multiplex Kit - 5 Color

Multiplex Fluorescence Immunohistochemistry (mIHC) Kit with TSA Amplification with 4 color fluorescence [520, 570, 620, 700] and DAPI. Compatible with mouse and rabbit primary antibodies.

IHC, mIHC, TMA

The fluorescent dye is provided as a 100× stock solution. To prepare the working solution, dilute the stock 1:100 with the freshly prepared signal amplification reaction solution. DAPI should be diluted 1:100 in sterile water to obtain its working solution. The kit also contains a universal secondary antibody compatible with mouse and rabbit primary antibodies; before use, verify that the primary antibody species matches the secondary antibody. The secondary antibody is supplied as a 1× working solution and is ready for use.

The multiplex fluorescence immunohistochemical assay is based on the specific binding between antigens and antibodies, combined with the principle of tyramide signal amplification (TSA). Similar to the DAB chromogenic reaction in conventional IHC, TSA technology utilizes horseradish peroxidase (HRP)-conjugated secondary antibodies. HRP catalyzes the conversion of a tyramide-linked fluorophore substrate into an activated intermediate that covalently binds to tyrosine residues within the antigen site, producing a stable and permanent fluorescent signal. After each labeling cycle, non-covalently bound antibodies are removed through heat-mediated antigen retrieval, allowing the application of a new primary antibody and a different fluorophore substrate in subsequent staining rounds. By repeating this process, multiple targets can be sequentially labeled within a single tissue section, enabling in situ multiplex detection and visualization of the complex cellular architecture of the tissue microenvironment with high spatial resolution.

The kit contains TSA monochromatic fluorescent dye 520, TSA monochromatic fluorescent dye 570, TSA monochromatic fluorescent dye 620, and TSA monochromatic fluorescent dye 700. These dyes emit green, yellow-orange, red, and far-red light respectively and are used with tyramide signal amplification to visualize multiple targets in the same sample. It also includes a signal amplification reaction solution, which provides the reagents necessary for the TSA reaction to deposit the fluorescent dye at the site of the target. A Pika universal HRP-labeled secondary antibody is included, which binds to the primary antibody and catalyzes the TSA reaction. The kit contains an anti-fluorescence quenching encapsulation tablet to protect the fluorescent signal from fading during imaging. Finally, DAPI is provided to stain cell nuclei and serve as a counterstain.

520 nm, 570 nm, 620 nm, 700 nm

Liquid

Shipped at +4 °C. Fluorescent dyes should be stored protected from light at +4 °C and are stable for up to one year from the date of receipt.

Microwave antigen retrieval conditions, incubation duration, and temperature can all significantly influence staining performance. Therefore, it is essential to follow the protocol precisely. Each staining run must include both a positive tissue control and a negative reagent control to ensure the validity and success of the procedure. For detailed instructions, always refer to the Product Manual included with the kit.