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.
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.