Depex mounting medium

Slides were baked for 60 min at 60 °C, deparaffinized with xylene for 5 min, rehydrated in ethanol (100%, 95%, 70%, and 50%). For antigen retrieval, slides were immersed in 10 mM sodium citrate and subjected to high heat and pressure for 20 min. After cooling, slides were rinsed with PBS for 10 min, and 3% H₂O₂ for 10 min. Slides were blocked in normal goat serum (ABC Kit, Vector Laboratories, Inc.) and 0.3%Triton x-100 in PBS and incubated overnight at 4 °C with primary antibodies: PPAR gamma (Bioss, catalogue no. bs-4590R, 1:200, Woburn, MA), PPARGC1A (Proteintech, catalogue no. 66369-1-Ig, 1:200, Rosemont, IL). YWHAZ (Bioss, catalogue no. bsm-215397M, 1:200, Woburn, MA), ZFYVE27 (Bioss, catalogue no. bs-11777R, 1:200, Woburn, MA). After PBS rinses, slides were incubated with biotinylated secondary antibodies diluted in the blocking solution for 2 hours at RT. The slides were developed using ImmPACT^TM DAB chromogen in ImmPACT^TM DAB diluent (ImmPACT^TM DAB peroxidase substrate kit, catalogue no. SK-410, Burlingame CA) and counterstained with hematoxylin. Slides were then dehydrated in ethanol (50%, 70%, 95%, 100%), immersed in xylene and coverslipped with DEPEX mounting medium (Electron microscopy sciences, cat# 13514, Hatfield, PA).

Brain sections were washed in 0.01 M phosphate buffered saline (PBS) for a total of 1 h (6 × 10 min), and then mounted on glass slides (Fisher Scientific Superfrost® Plus, Toronto, Canada) using 0.3% gelatin (Fisher Scientific, Toronto, Canada), and were dried overnight. Slides were incubated in 1% sodium hydroxide diluted in 80% ethanol for 5 min. They were then dehydrated in a graded series of ethanol (95% for 3 min, 70% for 3 min, and 50% for 2 min), washed in distilled water (3 × 1 min), and incubated in 0.06% potassium permanganate (Fisher Scientific) for 15 min on a shaker. The slides were washed with PBS (3 × 1 min) and placed in 0.0004% FluoroJade B (Chemicon, Etobicoke, Canada) for 20 min on shaker. Lastly, the slides were washed with PBS (3 × 1 min), dried in an incubator at 37°C for 25 min, cleared in xylene (Caledon Laboratories Ltd., Georgetown, Canada) for 1 min and coverslipped with Depex mounting medium (Electron Microscopy Sciences, Hatfield, USA).

The immunohistochemical staining of CSPG4 protein was performed as described before (18 (link)). Following the deparaffinization and rehydration of the tumor tissue, antigen retrieval was achieved by indirectly heating the tissue in a 1mM EDTA buffer with a pH of 8.0 for 15 minutes. To prevent non-specific binding we incubated the tissue microarray slide with a blocking buffer containing 1% Bovine Serum Albumin (BSA) and 5% normal horse serum (NHS) in Tris-Buffered Saline with Tween 20 (TBST). Incubation of the slides was performed overnight at 4 degree Celsius with the CSPG4-specific mAb D2.8.5-C4B8 (3 μg/mL) diluted in 1% BSA and 5% NHS in TBST. Subsequent staining was achieved with the DAKO EnVision+ System-HRP (Dako North America, Inc.) and substrate diaminobenzidine (Dako North America, Inc.). Counterstaining was performed with Mayer’s Hematoxylin (Lillie’s Modification, Dako North America, Inc.). Finally we mounted the slides with Depex mounting medium (Electron Microscopy Sciences ^®).
The staining intensity was graded by semiquantitative analysis by 2 investigators (SN, FS) where CSPG4 staining using IHC was scored using a categorical classification, 1-low expression; 2-medium expression; 3-high expression.