BRAIN RESEARCH ELSEVIER Brain Research 696 (1995) 49-61 Research report S-lOOfl has a neuronal localisation in the rat hindbrain revealed by an antigen retrieval method Qiner Yang a, Anders Hamberger a, Holger Hyden a, Shu Wang a, Torgny Stigbrand h, Kenneth G. Haglid a, * a Department of Anatomy and Cell Biology, University ofGbteborg, Medicinaregatan 5, 413 90 G6teborg, Sweden b Department of Medical Biochemistry and Biophysics, University of Ume?z, 901 87 Ume&, Sweden Accepted 31 May 1995 Abstract The localisation of S-100 in mammalian CNS neurons has been under debate for more than two decades. We address the question with two polyclonal and two new monoclonal antibodies. The specificity and the distribution in rat brain is based on an antigen retrieval method. We present evidence that aldehyde fixatives mask S-100/3 in neurons, and that the immunoreactivity is retrieved after trypsinisation. Neuronal S-100fl is also detected in unfixed and ethanol fixed sections. The neuronal immunoreactivity is partly solubilised from unfixed tissue sections with 2.5 mM EDTA and is completely extracted with 2.5 mM EDTA and 1% Triton X-100. Most of the glial S-100fl is washed out from unfixed tissue sections with saline. S-100/3 has distinct distribution in neurons of the hindbrain, i.e., the brainstem and cerebellum, but is not observed in the forebrain. One of the monoclonal antibodies immunostained neither neurons nor glia when it had been absorbed with S-100 crosslinked to nitrocellulose membranes. The distribution of neuronal S-100fl differed from that of other neuronal calcium binding proteins, such as calbindin and parvalbumin. It was confined mainly to cholinergic neurons of the hindbrain. The presence of S-100fl in distinct neuronal populations may indicate neurotrophic effects of S-100/3. The notion is supported by the capability of S-100 to cause neurite outgrowth in vitro. Keywords: Brainstem; Cerebellum; Immunocytochemistry; S-100fl; Trypsinization 1. Introduction S-100, a family of acidic calcium and zinc binding proteins, is localised mainly in astrocytes and a subpopula- tion of oligodendrocytes of the CNS [4,19,25,26,40,50], and in Schwann cells of the PNS [48,49]. However, S-100 is present also outside the nervous system [9,57]. The localisation of S-100 in CNS neurons is controversial. Its presence vs. absence was ascribed to technical differences and to the specificity of the antibodies [14,17,20,28,54]. Strong crosslinkers, such as formaldehyde and glutaralde- hyde, known to reduce S-100 immunoreactivity [12,31,47], are used in most studies on S-100 immunocytochemistry [4,8,14,28]. The localisation of S-100 is a prerequisite for the understanding of its function. The protein exists in three dimeric isoforms, S-100a0, S-100ot and S-100/3, formed * Corresponding author. Fax: (46) (31) 773-3330. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00755-5 by aa, aft and /3fl subunits, respectively. The distribu- tion of a and /3 subunits differs [20,23]. Both o~ and /3 are found in astrocytes, while ce is the only subunit in neurons [20,23]. No S-100a mRNA has been demon- strated in rat brain [24] and antibodies monospecific to S-100a labelled neither glia nor neurons [23]. Thus, the rat brain contains mostly S-100/3 [3]. The disulfide linked dimeric S-100/3 has been proposed to have a neurotrophic function [27]. S-100 regulates as- sembly/disassembly of the MAP2 and Tau proteins in vitro. However there is no colocalisation of these proteins [10]. The present study characterises the specificity and distribution of S-100/3 in neurons of the rat brain with well characterised polyclonal and S-100/3 specific mono- clonal antibodies. We report on results obtained with an antigen retrieval method in which paraformaldehyde fixed sections were trypsinised before the immunocytochemical processes. S-100fl immunoreactivity was observed, not only in astrocytes, but also in distinct neuronal popula- tions.