The challenge for immunohistochemistry is to choose the species of primary and secondary monoclonal antibodies to avoid cross-reactivity and obtain better specificity and repeatability. Gender decision is necessary to optimize the antibody dilution ratio. Meanwhile, another challenge is to align the localization of and the sub structures.

The use of this protocol meet the technical evolution of the distribution of three lymphocytic subpopulations, such as CT three, positive CT three, and CT eight positive and CT 20 positive based on in cell counts and the protein expression in conserv tissues, the level of infiltration can be observed too. In our protocol, the parameter Antibodies and the Fluor secondary antibodies derive from different species at the iso mixture making us a efficient. This measure also shows a specific measure between antibody and antigen.