Sections were deparaffinized and gradually hydrated in xylene and graded ethanol/distilled water solutions before performing antigen retrieval in 1X target retrieval solution (Dako, S1699) in a pressure chamber (SP1 Program, 125°C, 20 s; Pascal S2800, Dako). Samples were cooled to room temperature, treated with peroxidase-blocking solution (Dako, #S2023) for 1 h in a humid chamber and washed 3 times (5 min each) with PBST. Then, samples were blocked for 1 h in 5% normal horse serum (Vectastain Elite ABC HRP Kit R.T.U.; Vector laboratories, PK-7200) and incubated with specific primary antibodies for MYCN (dilution 1:100; Abcam, ab198912), CD31 (dilution 1:50, Abcam, ab28364), Ki67 (dilution 1:100, Abcam, ab15580), overnight at 4°C. After washing with PBST (5 min, 3 times), samples were incubated with secondary antibodies (Vectastain Elite ABC HRP Kit R.T.U.; 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.
Frozen sections of Neuroblastoma tumors were fixed in pre-cooled acetone (-20°C) for 10 min, washed with PBS, treated immediately with peroxidase-blocking solution and incubated with primary and secondary antibodies as explained above.
Frozen sections of Neuroblastoma tumors were fixed in pre-cooled acetone (-20°C) for 10 min, washed with PBS, treated immediately with peroxidase-blocking solution and incubated with primary and secondary antibodies as explained above.