Immunohistochemical staining for nerve fibers in normal tissues with frozen sections: Structure and Function Relationship

Abstract

Small nerve fibers are difficult to immunostain with routinely formalin-fixed and paraffin- embedded sections. Normal tissues except central nervous system are not densely innervated compared to densely distributed lymphatic and blood vessels and nerve fibers are not properly preserved in the routinely formalin-fixed and paraffin-embedded tissues. Nerve density technique with skin punch biopsy is an established method using 50 µm tissue sections, which are initially preserved in fixative. The fixed tissues are frozen sectioned and floating tissue sections are immunostained for nerve fiber markers. We had developed an alternative method using unfixed frozen sections mounted on glass slides. We had previously used this frozen section method for lymphatic and blood vessel immunohistochemistry and extended to nerve fiber immunostaining using neurofilament and CD56 as nerve fiber markers. Immunostained tissues with frozen sections were compared to formalin-fixed and paraffin-embedded colon and kidney sections. The normal tissues from rhesus monkey included heart, intestines, diaphragm, pancreas, spleen, kidney, thyroid, urinary bladder and others. Frozen section immunostained sections clearly depicted much more fine nerve fibers especially using CD56 as a nerve marker than with paraffin-embedded tissues. This technique is more labor-intensive but provides more precise distribution of fine nerve fibers than with routinely paraffin-embedded tissues. This immunostaining with frozen sections validated usefulness to depict more detailed nerve distribution and will shed light on normal histology and histopathology.