Immunofluorescence Labeling of Paraffin Tissue

For immunofluorescence labeling using paraffin embedded tissue sections the treatment procedure was performed as previously described^{8 (link),22 (link),88 (link)}. In sum, after deparaffinizing and buffering, the slides were incubated with a mixture of the rabbit IL-6 antibody (see above) and mouse anti-MAP2 (microtubule-associated protein 2, 1:200; Millipore, Temecula, USA) as well as mouse anti-GFAP (1:1000; Sigma-Aldrich, St. Louis, USA) in 5% fetal calf serum in buffer over night at 4 °C. The primary antibodies were visualized with Cy2-conjugated donkey anti-rabbit (1:400) and Cy5-conjugated donkey anti-mouse IgG (1:200; both Jackson ImmunoResearch, West Grove, USA) after 2 h incubation. Further, slides were stained with Hoechst 33342 (1:1000, Molecular Probes, Leiden, Netherlands) to identify the cell nuclei by auto-fluorescence utilizing an ultraviolet laser (362 nm). Strict internal control runs were done without primary antibodies.
The immunofluorescence was investigated by a confocal laser scanning microscope (Leica SP8 confocal microscope; Leica, Wetzlar, Germany) using excitation wavelengths of 488 nm (argon laser, yellow-green Cy2-immunofluorescence labelling), 543 nm (helium/neon1, red Cy3-immunofluorescence) and 633 nm (helium/neon2, blue Cy5-labelling).