Ab185924

To examine the capillary and arteriole densities, paraffin sections of Matrigel were stained with following antibodies: rat anti‐mouse CD31 (562939, BD Bioscience), rat anti‐rabbit luciferase (ab185924, Abcam) and rat anti‐rabbit GFP (ab290, Abcam). Alexa Fluor 488 or 550 conjugated antibody (Invitrogen) were used for secondary staining. After being mounted with Hochest mounting medium, the samples were analysed using a fluorescence confocal microscope (Leica). For morphometric analysis, sections were stained with haematoxylin and eosin (H & E). Images were taken under 200/400× magnification.

Mouse brains were harvested after formalin perfusion and cryopreserved using Optimal Cutting Temperature compound (Fisher Healthcare). Sections were cut 5-μm thick using Thermo Scientific Cryotome FSE cryostats. Tumors were confirmed using hematoxylin & eosin (H&E) staining, Ki67 (D3B5) (Cell Signaling Technology, #12202), H3.3K27M (D3B5T) (Cell Signaling Technology, #74829), and luciferase (Abcam, #ab185924) staining. Neurotoxicity was assessed via H&E and immunohistochemistry for glial fibrillary acidic protein (GFAP) (Agilent, #Z033429-2), Ionized calcium binding adaptor molecule 1 (Iba1) (Abcam, #ab5076), and cluster of differentiation 3 (CD3) (Abcam, # ab16669). Staining and immunohistochemistry were performed by the Weill Cornell Pathology Core. Slides were reviewed by D.J.P., a board-certified neuropathologist, for neurohistopathological changes observed in the treatment group against vehicle-treated animals.

Paraffin-embedded samples were sectioned at 4 μm thickness. Sections were de-paraffinized, rehydrated, and boiled in a pressure cooker for 2 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. Then sections were blocked in PBS containing 5% bovine serum albumin (BSA) for 15 min at room temperature. For immunofluorescence assay, sections were incubated with primary antibodies specific for firefly luciferase (Abcam, Cat# ab185924, 1:100) overnight at 4℃ and subsequently incubated with Alexa Fluor-488, 555, or 647 conjugated secondary antibodies (Thermo Fisher Scientific, Cat# A32731, A32727, A-21247, 1:300) for 1 hr at room temperature. Cells were counterstained with DAPI. Images were obtained by laser scanning confocal microscopy. For immunohistochemistry assay, sections were incubated with antibodies specific for luciferase (Abcam, Cat# ab185924, 1:100) overnight at 4℃, and later were detected by DAB (Dako) according to the manufacturer’s instructions.

All samples were resolved by SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in 0.1% PBS-T (phosphate buffered saline with Tween-20) and probed with an anti-Firefly antibody (ab185924, Abcam, 1/5000) or anti-SNAP-25 antibody (S9684, Sigma-Aldrich, 1/2000). Immunoreactive bands were detected using horseradish peroxidase–conjugated secondary anti-rabbit (A6154, Sigma-Aldrich, 1/2000) antibodies and SuperSignal West Dura ECL substrate (Thermo Fisher Scientific). Bands were visualized on a Pxi4 imaging system using GeneSys image acquisition software (Syngene, Bangalore, Karnataka, India).
For SNAP-25 cleavage assay, intensities of the total form and cleaved form of SNAP-25 were measured with GeneTools (Philomath, OR, USA).