Vectastainvr abc kit

Immunohistochemical studies were performed essentially as previously described [7 ]. Briefly, prior to immunostaining tissue underwent deparaffinization in xylene, followed by antigen retrieval, and blocking of endogenous peroxidase. The immunohistochemical staining was done using a standard labeled streptavidin-biotin method (VECTASTAINVR ABC Kit, # PK-4001, Vector Laboratories, CA, USA) followed by 3,3′-diaminobenzidine enzymatic development. The following antibodies were used: anti-PRMT1 antibody (Millipore, #07-404, 1:100 dilution) and anti-MYCN antibody (Santa Cruz, #sc-53993, 1:100 dilution). Normal rabbit or mouse IgG serves as the negative control. Slides were imaged with an Olympus VS120 slide scanner system. Immunoreactivity for PRMT1 and MYCN was independently scored by two investigators (JH and LL), both blinded as to the identity of each specimen, based on the previously described scoring method [7 ]. The statistical analysis was done by a two-tailed Fisher's exact test with a 95% confidence interval; P values < 0.05 were considered significant.

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.