Immunofluorescence experiments were performed as previously described [27 (link)]. Briefly, isolated tissues were fixed in 4% PFA solution at 4 °C overnight and then, after washing for 1 h in PBS, were immersed in 30% sucrose solution in PBS overnight. After washing in PBS, tissues were embedded in Tissue-Tek OCT compound (Sakura, The Netherlands) and frozen at − 80 °C. After blocking, sections were incubated with primary antibodies for 1 h at room temperature in blocking solution (0.1% Gelatin in PBS), washed for 30 min and then incubated with secondary antibodies for 1 h. Finally, the sections were washed for 15 min in PBS and mounted in PBS-glycerol (1:1) pH 8.0, containing 1% n-propyl gallate.
In the case of NMO-IgG staining, isolated tissues were frozen without fixation. Sections of 8 μm thickness were cut on a cryostat (CM 1900; Leica) at − 20 °C and stored on poly-l-lysine glass slides (positively charged) at − 80 °C. In this case, tissue sections were fixed at the end of the staining procedure to preserve conformational epitope integrity.
Finally, sections were viewed with a Leica DM2500 LED fluorescence microscope using 20X/0.55 and 40X/0.80 PL FLUOTAR objectives and photographed with a Leica DFC7000 T CCD camera. Confocal images were obtained using an automated inverted Leica TCS SP8 confocal microscope with a 100X HC PL Apo oil CS2 objective.
In the case of NMO-IgG staining, isolated tissues were frozen without fixation. Sections of 8 μm thickness were cut on a cryostat (CM 1900; Leica) at − 20 °C and stored on poly-l-lysine glass slides (positively charged) at − 80 °C. In this case, tissue sections were fixed at the end of the staining procedure to preserve conformational epitope integrity.
Finally, sections were viewed with a Leica DM2500 LED fluorescence microscope using 20X/0.55 and 40X/0.80 PL FLUOTAR objectives and photographed with a Leica DFC7000 T CCD camera. Confocal images were obtained using an automated inverted Leica TCS SP8 confocal microscope with a 100X HC PL Apo oil CS2 objective.