title: Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells.
abstract: Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900 W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.
authors.affiliation: Institute of Biological Sciences, Federal University of Triângulo Mineiro, Uberaba, MG, Brazil. armartin@fmrp.usp.br
authors.forename: Antonio R
authors.initials: AR
authors.lastname: Martins
authors.forename: Cesar A B
authors.initials: CA
authors.lastname: Zanella
authors.forename: Fabiola C R
authors.initials: FC
authors.lastname: Zucchi
authors.forename: Thaís C D
authors.initials: TC
authors.lastname: Dombroski
authors.forename: Edmar T
authors.initials: ET
authors.lastname: Costa
authors.forename: Liliane M
authors.initials: LM
authors.lastname: Guethe
authors.forename: Alina O
authors.initials: AO
authors.lastname: Oliveira
authors.forename: Ana L F
authors.initials: AL
authors.lastname: Donatti
authors.forename: Luciano
authors.initials: L
authors.lastname: Neder
authors.forename: Leila
authors.initials: L
authors.lastname: Chimelli
authors.forename: Gilberto
authors.initials: G
authors.lastname: De Nucci
authors.forename: Paulo
authors.initials: P
authors.lastname: Lee-Ho
authors.forename: Ferid
authors.initials: F
authors.lastname: Murad
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001160:Arterioles / Q000201:enzymology; D002460:Cell Line; D002525:Cerebellar Cortex / Q000201:enzymology*; D002675:Child, Preschool; D005260:Female; D005333:Fetus; D006099:Granuloma / Q000201:enzymology / Q000473:pathology; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008168:Lung / Q000166:cytology; D008297:Male; D008875:Middle Aged; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011180:Postmortem Changes; D020033:Protein Isoforms / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
chemical_list: D020033:Protein Isoforms; D019001:Nitric Oxide Synthase