Two-color immunoperoxidase staining: visualization of anatomic relationships between immunoreactive neural elements

A method for differentially staining for two antigens in single sections is described. Paraffin-embedded or Vibratome sections are incubated in two sequences of immunoperoxidase (PAP) reagents using a diaminobenzidine (DAB)-nickel ammonium sulfate solution to localize the first antigen, and DAB alone to localize the second antigen. With these chromagens, black and amber-colored reaction products are generated at the locations of the first and second antigens, respectively. The reaction products are stable and provide excellent color contrast. With this technique, the anatomical relationships between two sets of immunoreactive elements can be studied in individual sections. Intimate spatial associations that would probably not be detected by an examination of adjacent sections stained for each antigen can be visualized with this two-color immunoperoxidase method.