ABSTRACT
Immunostaining techniques are widely used for co-localization studies of multiple peptide antigens. In this chapter different immunohistochemical staining methods are reviewed and their advantages and disadvantages are discussed. Single immunostaining on consecutive sections is briefly evaluated. Particular attention is paid to methods for double or triple immunostaining on the same section, both for brightfield light microscopy (elution and nonelution techniques) and fluorescence microscopy (including methods to avoid cross-reactivity of primary and secondary antibodies). Various fluorophore properties that may influence their choice in a given staining procedure are commented. In addition, methods for quadruple immunostaining on the same section are mentioned. For co-localization studies, multiple immunostaining using fluorophores usually have more advantages than the other methods, and protocols are suggested in Section 1.5.
