Anti nrp1 antibody

Cells were lysed in RIPA buffer, and the cell lysates were treated with PNGase F (peptide N-glycosidase F) for 3 h at 37 °C according to the manufacturer’s instructions (#P0704, New England Biolabs). The reaction was stopped by the addition of 5 × loading buffer followed by western blot analysis using anti-NRP1 antibody (#3725, Cell Signaling Technology).

5 μm sections of FFPE medulloblastoma tissues were deparaffinized, rehydrated and the antigen retrieval was done as per the protocol (http://www.cellsignal.com). Non-specific binding to the sections was blocked using 3% Bovine serum albumin (BSA) for 1 h. The sections were incubated in 1:100 diluted anti-NRP1 antibody (Cell signaling technologies, Boston, MA, USA) in 1% BSA overnight at 4ºC, followed by incubation with the peroxidase conjugated secondary anti-rabbit IgG (Pierce, Thermo Fischer scientific, Waltham, MA, USA). As a negative control, the sections were treated identically but without the primary antibody. The bound peroxidase signal was detected using 3, 3-' Diaminobenzidine as a substrate. The staining was visualized and photographed using Zeiss Upright fluorescent microscope Axioimager. Z1 and scored on the basis of the intensity and percentage of positive cells. The scoring of the staining intensity by a neuropathologist blinded to the molecular subgroup or survival data.