Immunohistochemical visualization of neurons and specific glial cells for stereological application in the porcine neocortex

The pig is becoming an increasingly used non-primate model in basic experimental studies of human neurological diseases. In spite of the widespread use of immunohistochemistry and cell type specific markers, the application of immunohistochemistry in the pig brain has not been systematically described. Therefore, to facilitate future stereological studies of the neuronal and glial cell populations in experimental neurological diseases in the pig, we established a battery of immunohistochemical protocols for staining of perfusion fixed porcine brain tissue processed as free floating cryostat-, vibratome- or paraffin sections. Antibodies against NeuN, GFAP, S100-protein, MBP, CNPase, CD11b, CD68 (KP1), CD45 and Ki67 were evaluated, and all except CD68 and CD45 resulted in staining of high quality in either type of tissue. Each staining was evaluated with respect to specificity and sensitivity in identification of the individual cells, and for penetration of the staining and maintenance of section thickness above 25 μm, necessary for stereological cell counting. In the cases of NeuN, CNPase, CD11b and Ki67 the staining met the demands to be applicable in stereological analyses using the optical disector. In conclusion, all protocols will be applicable in studies of pathological and neurochemical changes in the porcine brain, and a few protocols applicable for stereology.