Fig. 2

Representative figures of IHC for polyglutamylation. a GBM specimen used as positive control and stained for polyglutamylation (PG). Specimens of meningioma and pituitary adenoma, and the GBM specimen without primary antibodies were used as negative controls, revealing no staining for PG. Magnification, 200×. b Each subset of panels (left and right) shows polyglutamylated and non- polyglutamylated tumor specimens, respectively. The left upper panels are stained with hematoxylin and eosin, and the right upper panels show IHC results for CD20. Magnification, 200×. The bottom panels show IHC results for PG. Magnification, 400×. c Histogram showing the percentage of stained cells (10% cut-off). d Each subset of panels (upper and lower) shows polyglutamylated and non-polyglutamylated tumor specimens, respectively. Multiple-fluorescence staining using two different antibodies was performed. PG (red; left), CD20 (green; middle), and the merged fluorescence image (right), including DAPI (blue). After subtraction of background of autofluorescence, double-positive staining for both PG and CD20 revealed cytoplasm with a yellow-to-orange color as a result of overlapping fluorescence in the merged image in the polyglutamylated group. The red-fluorescent region (lower right) in the merged image of the polyglutamylated tumor specimens was considered brain parenchyma