RESUMO
Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample's autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.
Assuntos
Anticorpos/análise , Imunofluorescência/métodos , Microscopia Imunoeletrônica/métodos , Animais , Anticorpos/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Lead exposure induces DNA damage, oxidative stress, and apoptosis, and alters DNA repair. We investigated the effects of melatonin co-administered to rats during exposure to lead. Three doses of lead acetate (10, 50 and 100mg/kg/day) were administered to rats during a 6-week period. Lymphocytes were analyzed. Lead exposure decreased glutathione (GSH) levels in blood, and at doses of 100mg/kg/day and 50mg/kg/day without melatonin, caused high levels of DNA damage, induced apoptosis, and altered DNA repair. Melatonin co-treatment did not attenuate the effects of lead at 100mg/kg/day, indicating that the effect of melatonin on GSH reduction is not sufficient to reduce the genotoxic effects of lead at this high dose. After 6 weeks of treatment, decreased weight gain was observed in high lead-dose groups (100mg/kg/day), with or without melatonin, and in medium-dose groups (50mg/kg/day) with melatonin, compared with the control group. The protective action of melatonin against lead toxicity is dependent on the dose of lead. Further pharmacological studies are needed to determine whether melatonin acts via melatonin membrane receptors on lymphocytes.
Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Melatonina/farmacologia , Compostos Organometálicos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Glutationa Peroxidase/metabolismo , Linfócitos/metabolismo , Compostos Organometálicos/administração & dosagem , Ratos , Ratos WistarRESUMO
La inhalación de disolventes industriales es uno de los problemas de salud más importantes tanto en medicina del trabajo, como en la esfera de la farmacodependencia cuando estas substancias son inhaladas involuntaria o voluntariamente. La inhalación voluntaria ha crecido alarmantemente en las ultimas décadas, convirtiendola en un objeto de estudio de importancia primaria, tanto por sus orígenes como por su magnitud. Los estudios pioneros de Costero y Barriso entre otros dan una idea de la magnitud de algunos de los efectos, que tiene esta farmacodependencia. Este trabajo se enfoca al estudio del efecto de la inhalación de tíner por periodos prolongados tienen sobre la morfología y la función del hígaso de la rata y la fracción mitocondrial aislada de ese órgano.