Use of fixatives for immunohistochemistry and their application for detection of retinoic acid synthesizing enzymes in the central nervous system

Abstract

Retinoic acid (RA) is a lipid signaling molecule that has a crucial role in growth and survival of neurons as well as regulation of neuronal plasticity in the central nervous system. Complete understanding of the distribution of RA is necessary to identify foci of RA signaling. However, RA itself is very difficult to detect by immunohistochemistry as there are few effective antibodies to this lipid and it can be difficult to fix in place in tissue. A set of retinaldehyde dehydrogenases (RALDHs) catalyze the last step of RA synthesis and their distribution can be used as a proxy for RA distribution. This protocol uses the example of RALDH2 expression in the motor neurons of the spinal cord as a demonstration of the approach and describes methods that can improve its effectiveness. Immunodetection is impacted by protein cross linking and protein denaturation as well as antigen/epitope masking by various fixatives. Finding a suitable fixative that preserves morphology and tissue structure by linking cellular component such as proteins and lipids in an indissoluble macromolecular network while keeping functional groups, including antigens, intact is essential. Here, we discuss fixatives in general and also describe a fixation protocol that allows detection of multiple antigens in the same section with a periodate-lysine-paraformaldehyde (PLP) fixative. This keeps tissue structure and antigen well preserved in the adult spinal cord to show RALDH2 expression in motor neurons.

Keywords: Acetone/ethanol; Antigen; Coagulating and cross-linking fixatives; Motor neurons; Periodate-lysine-paraformaldehyde; Retinaldehyde dehydrogenase 2; Spinal cord; Vitamin A.