Nanozoomer 2 ors

Immunostaining was performed at the time of initial diagnosis. Representative zinc formalin-fixed sections were deparaffinized and were subject to a Ventana autostainer (BenchMark XT, Ventana Medical Systems or Discovery XT, Ventana Medical Systems) according to standard protocol. The following primary antibodies were used: Glial Fibrillary Acidic Protein (GFAP) (1:200, 6 F2, Dako Denmark A/S, Glostrup, Denmark), synaptophysin (1:20, SY38, Progen Biotechnik GmbH, Heidelberg, Germany), CD34 (1:40, QBEnd-10, Dako, Denmark A/S, Glostrup, Denmark), chromogranin A (1:200, LK2H10, Diagnostic BioSystems, Pleasanton, USA), phospho-FGFR1 (Y653/654) (1:75, PA5-12594, Thermoscientific, Waltham, USA), p53 (1:5000, DO-1, Santa Cruz Biotechnology, Dallas, USA), BRAF V600E (1:100, VE1, Spring Bioscience, Pleasanton, USA), histone H3.3 K27M mutation (1:1000, ABE419, EMD Millipore, Billercia, USA). The chromogen diaminobenzidine was employed. Slide scanning was performed using NanoZoomer 2.ORS (Hamamatsu photonics, Hamamatsu, Japan).

The length of immunolabelling of Caspr/paranodin aggregates on cryostat or paraffin sections was measured in the cerebellum and internal capsule. The measurements were performed on images obtained by NanoZoomer 2.O-RS (Hamamatsu) apotome equipped with 20X and 40X objectives or a Leica DM400B optical microscope equipped with 40X, 63X and 100X objectives, according to the chromogen used, respectively fluoroscein, CY3 or DAB. Images acquired from epifluorescence or light microscope were superimposed using ImageJ. The length of paranodin immunostaining was measured inside and outside the plaque in affected and control tissues; the lengths were compared using a variance analysis with Anova.

Paraffin-embedded tissue was cut at 4 µm, mounted on slides and dried at 58 °C for 60 min. Hematoxylin Eosin Safran (HES) staining was performed to reveal the architecture of the spleen. The slides were scanned using a scanner (Nanozoomer 2.ORS; Hamamatsu, Massy, France) and areas of germinal centers (GC) were quantified using the NDP view 2.4.26® software (Hamamatsu).