Double immunofluorescence and confocal microscopy

The treatment procedure for immunofluorescence labeling was performed as described previously [23].

Briefly, the primary antibodies used were polyclonal rabbit anti-HSP70 (Zytomed Systems), mouse anti- microtubule associated protein-2 (MAP2, 1:200; Merck Millipore, Billerica, USA) and mouse anti-glial fibrillary acidic protein (GFAP, 1:1000; Sigma-Aldrich, St. Louis, USA). The simultaneous visualization of different primary antisera was performed with a mixture of secondary antibodies specific to the appropriate species IgG (rabbit, mouse): Carbocyanine (Cy)2- (1:400) and Cy5- (1:100) conjugated IgGs (all Jackson ImmunoResearch, West Grove, USA). Control experiments were performed without primary antibodies.

The multiple immunofluorescence was investigated by a confocal laser scanning microscope (LSM 510 Meta, Zeiss; Oberkochen, Germany) using excitation wavelengths of 488 nm (argon, yellow-green Cy2-immunofluorescence), 543 nm (helium/neon1, red fluorescence) and 633 nm (helium/neon2, blue Cy5-immunofluorescence).

Cy3-labeled antibodies were not used in this study, because of the unspecific auto-fluorescence of lipofuscin with an excitation wavelength of 543 nm (helium/neon1, red fluorescence). Therefore, this wavelength was used as a control for the presence of unspecific lipofuscin autofluorescence in the cells of interest. All red immunofluorescence labeling of different structures shown in the figures prepared resulted from color coding of the Cy5 immunofluorescence to allow a better differentiation between the labeled cells.