Clone y69

Immunostaining for MYCN and MYC protein was performed using formalin-fixed, paraffin-embedded sections with Leica BOND-MAXTM (Leica Microsystems Inc., Bannockburn, IL, USA) heating for 30 min in BondTM Epitope Retrieval Solution 2 (No. AR9640; Leica Biosystems Newcastle Ltd., Benton Ln, Newcastle Upon Tyne, UK). The sections were incubated with either anti-MYCN mouse monoclonal antibody, NCM II 100 (Ikegaki et al, 1986 (link)) at a dilution of 1 : 200, or anti-human MYC rabbit monoclonal antibody, clone Y69 (No. 1472-1; Epitomics, Cambridge, MA, USA) (Kluk et al, 2012 (link)) at a dilution of 1 : 200 in Bond TM Primary Antibody Diluent (No. AR9352; Vision BioSystems Inc., Norwell, MA, USA). Staining was visualised using Bond Polymer Refine DetectionTM (No. DS9800; Leica Microsystems Inc., Bannockburn, IL, USA). The slides stained for MYC protein were counterstained with hematoxylin. No counterstaining was performed for the slides after MYCN protein staining. The slides were reviewed by HS, LLW, and RT, and results were graded as follows: negative (−); focal or sporadic, and weak nuclear staining (±); and diffuse and strong nuclear staining with typically heterogeneous intensity (+). Appropriate positive and negative controls were stained along with those tumours.