Immunostaining for NeuN Does Not Show all Mature and Healthy Neurons in the Human and Pig Brain: Focus on the Hippocampus.

Neuronal nuclei (NeuN) is a neuron-specific nuclear protein, reported to be stably expressed in most postmitotic neurons of the vertebrate nervous system. Reduced staining has been interpreted by some to indicate loss of cell viability in human studies, while others suggest this may be because of changes in the antigenicity of the target epitope. Preliminary studies in our laboratory found low immunostaining for the NeuN antibody on formalin fixed and paraffin embedded (FFPE) human brain tissue. We report on the techniques and results used to enhance the staining for NeuN in that tissue. In parallel, we stained NeuN in piglet brain tissue, sourced from an experimental model where methodological parameters, including those for tissue fixation and storage, were tightly controlled. In human FFPE brain tissue, we were unable to enhance NeuN immunostaining to a degree sufficient for cell counting. In contrast, we found consistently high levels of staining in the piglet brain tissue. We conclude that processes used for fixation and storage of human FFPE brain tissue are responsible for the reduced staining. These results emphasize that a cautionary approach should be taken when interpreting NeuN staining outcomes in human FFPE brain tissue.