Fv 300 confocal fluorescence microscope

After transduction of SAG and Tat-SAG into SH-SY5Y cells, their intracellular distributions were assessed by confocal fluorescence microscopy. Cells were grown on glass coverslips and exposed to SAG and Tat-SAG (3 μM) for 60 min. The cells were washed with PBS three times and fixed with 4% paraformaldehyde at room temperature for 10 min, followed by incubation with an anti-histidine antibody and Alexa fluor 488-conjugated secondary antibody (Invitrogen, USA). 4′6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. The fluorescence image was analyzed using an Olympus FV-300 confocal fluorescence microscope.

For detection of transduced PEP-1-Catalase in SH-SY5Y cells, Confocal fluorescence microscopy was performed as described previously (10 (link), 13) (link). The cells were seeded on coverslips after which they were exposed to PEP-1-Catalase and control catalase protein (3 μM) for 3 h. Cells were then washed with PBS twice and fixed with 4% paraformaldehyde at room temperature for 5 min. The cells were incubated with an anti-histidine primary antibody and an Alexa Fluor 488-conjugated secondary antibody (Invitrogen; Carlsbad, CA, USA). Nuclei were stained for 5 min with 1 μg/ml 4'6-diamidino-2-phenylindole (DAPI; Roche Applied Science, Basel, Switzerland). An Olympus FV-300 confocal fluorescence microscope (Olympus, Tokyo, Japan) was used to analyze fluorescence images.