Validating immunofluorescence methodologies for neuroanatomical studies of the Substantia Nigra.

Abstract

Aims: Define methodologies and reagents for neuroanatomical studies on the Substantia Nigra (SN). Background: Antibodies are valuable neuroscience research tools which require careful characterization prior to their utility in answering specific research questions (1). In adopting methodologies and antibodies for specific studies, the conservation of antigens, cellular structure and specificity of the antibodies need defining. Here we test and optimize immunofluorescence methodologies for studies on the rat SN with specific relevance to studies on human brain tissues. Methods: Adult male Wistar rats (250-300g) were euthanised (i.p. lethabarb), stored at 4ºC and brains harvested at 0, 2, 4, 8, 16 and 24 hours post-mortem (n=4 for each timepoint), then immersion fixed overnight at room temperature in 4% paraformaldehyde. Brains were bisected midline sagitally and one half processed for paraffin and the other for polyethylene glycol embedded sectioning. Four rats were perfusion fixed transcardially and brains processed as above, for positive controls. Perivascular sympathetic nerves in human tissues were used for testing cross species specificity of tyrosine hydroxylase (TH) antibodies (4 antibodies). Cross reactivity of TH antisera with tryptophan hydroxylase (TPH) was tested in rat raphe nucleus. Antibodies against serotonin (5-HT), Girk-2 and calbindin were used to further validate the results. Conclusions: Increasing postmortem duration and paraffin embedding protocol resulted in reduction of TH antigenicity, which was cumulative. All TH-antibodies showed affinity for rodent and human antigen, however, three cross reacted with TPH, with only one being specific for TH. Here we define findings especially relevant for studies on human cadaver tissues. 1. Rhodes, K.J. and J.S. Trimmer, Antibodies as valuable neuroscience research tools versus reagents of mass distraction. J Neurosci, 26(31): 8017-8020 (2006).