Anti eya1 antibody

Immunohistochemical studies were performed essentially as previously described [22 ]. Briefly, prior to immunostaining tissue underwent deparaffinization in xylene, followed by antigen retrieval in citrate buffer, and blocking of endogenous peroxidase. The immunohistochemical staining was done using a standard labelled streptavidin-biotin method (VECTASTAINVR ABC Kit, # PK-4001, Vector Laboratories, CA, USA) followed by 3,3′-diaminobenzidine enzymatic development. The nonspecific binding was blocked by incubating with goat serum and Avidin D/Biotin. The sections were incubated with anti-EYA1 antibody (Prosci, #25-067, 1:100 dilution) or anti-MYCN antibody (Santa Cruz, #sc-53993, 1:100 dilution) for 30 min and then goat anti-rabbit or anti-mouse biotinylated IgG for 30 min, followed by incubation with ABC reagents. Hematoxylin was used for nuclear counterstaining. Slides were dehydrated through graded alcohol and xylene washing, and mounted on cover slips. Normal rabbit or mouse IgG served as the negative control. Slides were imaged with an Olympus VS120 slide scanner system.

KELLY cells were purchased from Sigma (St. Louis, MO) and cultured in DMEM:F12, supplemented with 10% FBS. HEK293TN cells were cultured in DMEM with 10% FBS. Transient transfection was performed by using jetPRIME (Polyplus transfection, #114-07) per the manufacturer’s instructions. CoIP analysis was performed essentially as previously described[21 (link)]. Briefly, two days post-transfection, whole cell lysates of HEK293TN cells were prepared and used for V5-IP (Invitrogen, #R960-25). In the case of KELLY cells, nuclear extracts were isolated and subjected to immunoprecipitation with anti-MYCN antibody (Santa Cruz, #sc-53993), followed by immunoblotting with anti-EYA1 antibody (Prosci, #25-067). The following antibodies were used in western blot: α-Flag (Thermo Fisher Scientific, #MA1-91878) and β-actin (Sigma, #A5316).