Triple enzymatic immunochemistry for interneuron populations in postmortem human cerebral cortex

Immunostaining is an antibody-based tool used to visualize proteins in tissue. Enzymes or fluorochromes conjugated to antibodies are used to detect proteins of interests. Fluorescent immunostaining can be used in human tissue, however due to the high autofluorescence of non-perfused human tissue, enzymatic immunostaining is better suited. Enzymes produce a colored product that is detectable by light microscopes. Here we describe a successful triple immunochemistry protocol to enzymatically label three distinct populations of interneurons (Parvalbumin+, Calbindin+, and Calretinin + interneurons) in non-perfused formalin fixed human brain cerebral cortex. Signal was achieved using a combination of horseradish peroxidase (HRP) and Alkaline Phosphatase (AP) enzymes and color was generated using the insoluble chromogens: 3,3′- Diaminobenzidine (DAB, Brown), Vector Blue (Blue), and Vector VIP (Pink). There were no noticeable background and minimal signal overlap between the different colors. We were able to successfully stain human cortical tissue and distinguish morphological properties of the three interneuron (IN) populations.