Impact dab

Frozen sections of the native NB tumors were fixed in pre-cooled acetone (−20°C) for 10 min, washed with PBS and treated with 0.3% H₂O₂ solution in PBS at room temperature for 10 min to block endogenous peroxidase activity. The samples were blocked for 1 h in 5% normal horse serum (Vectastain Elite ABC HRP Kit R.T.U.; Vector laboratories, PK-7200). The samples were then incubated with the following primary antibodies diluted in antibody diluent (Dako, S3022), in a humid chamber overnight at 4°C: Collagen 1 (dilution 1:500; Abcam, ab34710), EZH2 (dilution 1:50; Millipore # 07–689) and GLI1 (GLI1-rabbit dilution 1:50, Cell Signaling, #2553). The next day, the sections were washed (3 times, 5 min each) with PBST and incubated with secondary antibodies (Vectastain Elite ABC HRP Kit R.T.U. from Vector laboratories, PK-7200), following manufacturer instructions, and developed using Impact DAB (Vector Laboratories, SK4105). Negative controls were prepared by omitting the primary antibody step. Slides were counterstained with Hematoxylin QS (Vector Labs).
For the hyaluronan acid binding protein (HABP) staining, the sections were blocked using 1% BSA in HBSS at room temperature for 30 min, and incubated with a biotinylated HABP antibody (dilution 1:100; Millipore #385911). A Streptavidin Alexa Fluor 488 conjugate (dilution 1:500, Molecular Probes) was used as a secondary antibody.

Reactive astrogliosis was assessed by quantification of glial fibrillary acidic protein (GFAP). Tissue sections were washed in PBS and endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol for 10 minutes. Sections were then blocked with a solution of 2% goat serum and 0.2% Tween 20 in PBS for one hour and then incubated in blocking buffer overnight at 4°C with a primary antibody against GFAP (ab4674, Abcam, Cambridge, UK; 1:8000). They were then rinsed with PBS and incubated with a biotinylated secondary antibody (Vector Labs, Burlingam, CA; 1:200; 1 h). Immunolabeling was visualized with VECTASTAIN ABC and Impact DAB (Vector Labs). Using SPOT Basic imaging software (Sterling Heights, MI), the dorsal striatum was photographed at 10× magnification across 4–6 coronal sections from each subject (n = 7/group). The camera frame was centered within the dorsal striatum, and GFAP immunoreactivity was quantified within the frame using ImageJ by an observer blinded to the treatment condition. Images were converted to 8-bit, and contrast and brightness were adjusted equally for all images using the default settings (imagej.nih.gov/ij/plugins/plane-brightness). Artifacts were removed if needed, and the percent area of immunolabeling was determined by analyzing the particle area with a threshold set to include only immunolabeled cells.