Morawski M, Brückner G, Jäger C, Seeger G, Arendt T. Neurons associated with aggrecan-based perineuronal nets are protected against tau pathology in subcortical regions in Alzheimer's disease. Neuroscience. 2010 Sep 1;169(3):1347-63. PubMed Abstract

REAGENTS/MATERIAL:
Detection of extracellular matrix components: Sections were incubated with rat monoclonal anti-aggrecan ( HAG7D4) (Acris, Hiddenhausen) raised against human cartilage aggrecan. The antibody detects the N-terminal part of the aggrecan core protein in brain tissue of different mammalian species and human as shown in various studies.
 
Detection of neurotransmitter systems: The rabbit anti-tyrosine hydroxylase (Chemicon, now Millipore) raised against denatured TH from rat pheochromocytoma was used to label dopaminergic and noradrenergic neurons. The antibody recognizes an epitope in the midportion of the TH molecule, where extensive species homology exists and, thus, has wide species cross-reactivity.
The affinity-purified goat polyclonal anti-choline acetyltransferase (ChAT) (Millipore) against human placental ChAT was used to label cholinergic neurons.
The affinity-purified goat polyclonal anti-orexin-A (Santa Cruz, sc-8070) was raised against a peptide mapping at the C-terminus of orexin-A of human origin.
The mouse monoclonal anti-tryptophan hydroxylase was derived from the WH-3 hybridoma cells and was produced by the fusion of mouse myeloma cells and splenocytes from BALB/c mice. Recombinant rabbit tryptophan hydroxylase was used as immunogen. The antibody specifically labels tryptophan hydroxylase in the mammalian nervous tissue (Washburn et al., 2002).
The affinity-purified goat polyclonal anti-urocortin (Santa Cruz, sc-1825) was raised against a peptide mapping at the C-terminus of urocortin of rat origin. The antibody specifically labels urocortin in mammalian nervous tissue.
 
Detection of tau-pathology:. Cytoskeletal pathology related to hyperphosphorylation and aggregation of the tau protein sections was detected by a variety of well characterized antibodies. The affinity-purified rabbit polyclonal anti-tau phosphospecific antibody (Invitrogen, pT205) was raised against a chemically synthesized phosphopeptide derived from the region of human tau that contains threonine 205.
The mouse monoclonal anti-PHF-tau (Thermo Scientific, clone AT8) recognizes tau in its native state of phosphorylation and does not detect the six human isoforms of unphosphorylated tau. It is specific for PHF-tau phosphorylated both at Ser202 and Thr205. It has also been shown to cross-react with tau phosphorylated both at Ser199/202 and Ser205/208.
To assure that the tau immunoreactivity is not masked by dense DAB-Ni reaction product randomly selected sections were immunostained with double-fluorescent labelling by pT205 antibody and aggrecan antibody.