title: The tungstate-stabilized tetramethylbenzidine reaction for light and electron microscopic immunocytochemistry and for revealing biocytin-filled neurons.
abstract: A peroxidase reaction product that can be easily distinguished from standard diaminobenzidine (DAB) reaction products is needed for pre-embedding electron microscopic double-antibody labelling studies. Benzidine dihydrochloride (BDHC) and gold-substituted silver peroxidase reactions are unsatisfactory for double labelling because they lack sensitivity and reliability and/or compromise ultrastructure. We show here that light and electron microscopic immunocytochemistry can be done with a modification of the tungstate-stabilized tetramethylbenzidine (TMB) reaction (Weinberg and Van Eyck 1991) which yields a crystalline reaction product. With this method, we have obtained excellent immunolabelling for a variety of antigens, including tyrosine hydroxylase, enkephalin, serotonin, Fos protein and retrogradely transported cholera toxin B subunit (CTB). The TMB-tungstate reaction is useful for ultrastructural double labelling because the crystals contrast well with the amorphous product of diaminobenzidine reactions. The TMB-tungstate reaction is more sensitive and reliable for immunocytochemistry than the benzidine dihydrochloride reaction and gives better ultrastructure than the gold-substituted silver peroxidase reaction. We also show that neurons filled with biocytin by intracellular injection can be visualized with TMB-tungstate for either light (LM) or electron (EM) microscopy.
authors.affiliation: Centre for Neuroscience, School of Medicine, Flinders University, South Australia.
authors.forename: I J
authors.initials: IJ
authors.lastname: Llewellyn-Smith
authors.forename: P
authors.initials: P
authors.lastname: Pilowsky
authors.forename: J B
authors.initials: JB
authors.lastname: Minson
mesh_terms: D000818:Animals; D001560:Benzidines / Q000737:chemistry*; D003460:Crystallization; D004594:Electrophysiology; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000648:ultrastructure; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry / Q000379:methods*; D008239:Lysine / Q000031:analogs & derivatives*; D008854:Microscopy, Electron; D009474:Neurons / Q000648:ultrastructure*; D009532:Nickel / Q000737:chemistry; D051381:Rats; D011921:Rats, Inbred WKY; D013116:Spinal Cord / Q000166:cytology / Q000648:ultrastructure; D016707:Tissue Fixation; D014414:Tungsten / Q000737:chemistry*; D017973:Tungsten Compounds
chemical_list: D001560:Benzidines; D017973:Tungsten Compounds; C021758:3,3',5,5'-tetramethylbenzidine; D009532:Nickel; C013411:biocytin; D008239:Lysine; C045951:tungstate; D014414:Tungsten