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Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas.
Ninfali, P; Biagiotti, E; Guidi, L; Malatesta, M; Gazzanelli, G; Del Grande, P.
Afiliação
  • Ninfali P; Istituto di Chimica Biologica "G. Fornaini", University of Urbino, Via A. Saffi 2, I-61029, Urbino, Italy. p.ninfali@bib.uniurb.it
Brain Res Brain Res Protoc ; 5(1): 115-20, 2000 Feb.
Article em En | MEDLINE | ID: mdl-10719273
This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO(4), dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas.
Assuntos
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Bulbo Olfatório / Córtex Cerebral / Microscopia Imunoeletrônica / Glucosefosfato Desidrogenase Tipo de estudo: Diagnostic_studies Limite: Animals Idioma: En Ano de publicação: 2000 Tipo de documento: Article
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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Bulbo Olfatório / Córtex Cerebral / Microscopia Imunoeletrônica / Glucosefosfato Desidrogenase Tipo de estudo: Diagnostic_studies Limite: Animals Idioma: En Ano de publicação: 2000 Tipo de documento: Article