RESUMEN
Immunofluorescence is the basis for many techniques used to quantify phenomena in neuroscience research, in both normal and pathological tissue. Autofluorescence (non-specific, broad spectrum background fluorescence) is an unfortunate consequence of damage to brain tissue. Damage-induced autofluorescence potentially confounds analyses of tissue labeled with fluorescent markers in many experiments. This is especially problematic in protocols that utilize co-localization methods such as BrdU/NeuN in which autofluorescence might lead to overestimates of the number of double-labeled cells. Techniques to reduce autofluorescence are variable and relatively ineffective in damaged brain tissue. Here we show using confocal microscopy that damage-induced autofluorescence does not co-localize with the nuclear specific markers DAPI or Hoechst. Further co-localization of nuclear markers such as Ki67 or BrdU/NeuN with DAPI or Hoechst should serve to help discriminate between intended and spurious fluorescent signal.
Asunto(s)
Anticuerpos/análisis , Infarto Encefálico/metabolismo , Núcleo Celular/metabolismo , Fluorescencia , Microscopía Confocal/métodos , Neuronas/metabolismo , Animales , Anticuerpos/metabolismo , Artefactos , Bencimidazoles/análisis , Bencimidazoles/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Infarto Encefálico/diagnóstico , Infarto Encefálico/fisiopatología , Núcleo Celular/patología , Giro Dentado/metabolismo , Giro Dentado/patología , Modelos Animales de Enfermedad , Reacciones Falso Positivas , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Indoles/análisis , Indoles/metabolismo , Antígeno Ki-67/análisis , Antígeno Ki-67/metabolismo , Masculino , Neuronas/patología , Valor Predictivo de las Pruebas , Ratas , Ratas Long-Evans , Sensibilidad y EspecificidadRESUMEN
Two envelope fusion protein gene homologues have been identified in the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV GP64 protein is fusogenic and essential for propagation and pathogenicity. The F homologue (Ac23) is not essential, is fusion-incompetent in standard assays, but contributes to faster host death. Here, we show that occlusion bodies (OBs) from Ac23null mutants and control viruses do not differ significantly in size and the number of occlusion-derived virions (ODVs) contained; however, Ac23null OBs had a much higher percentage of ODVs with a single nucleocapsid (44.6%) than the near-isogenic control (11.3%). Infection of Sf9 cells with Ac23-green fluorescent protein (gfp)-expressing recombinant viruses showed Ac23-gfp fluorescence overlapping perinuclear DAPI staining at later times, a pattern not observed with GP64. These results suggest that F proteins have evolved functions beyond envelope fusion and play a different role from that of GP64 in viruses that contain both proteins.
Asunto(s)
Núcleo Celular/virología , Nucleopoliedrovirus/fisiología , Proteínas Virales/fisiología , Ensamble de Virus , Replicación Viral , Animales , Línea Celular , Eliminación de Gen , Nucleopoliedrovirus/genética , Spodoptera , Proteínas Virales/genéticaRESUMEN
The main objective of the present study was to perform an unbiased comparison of immersion vs. perfusion techniques to assess whether we could use the former to quantify synapses through electron microscopy (EM). Using the immersion technique is ideally suited for instances in which the specimen under study could not be perfused under the standard EM protocol. Our results suggest that, despite suboptimal qualitative results, fixation by immersion allows for adequate quantification of synapses.