Immunohistochemical (IHC) staining was performed on paraffin sections of
neuroblastoma tissue microarray assay (TMA) and xenograft tumors. Paraffin
sections were deparaffinized, and antigen retrieval was carried out in citric
buffer in microwave for 10 min. The sections were incubated in 1%
hydrogen peroxidase for 10 minutes to quench endogenous tissue peroxidase.
Tissue sections were then incubated with the anti-GSK-3β antibody (Cell
Signaling, Danvers, MA) overnight at +4°C. The slides were stained using
a standard EnVision+ System-HRP kit (Dako, Carpinteria, CA) according to the
manufacture’s protocol. Immunohistochemical reactions were developed
with diaminobenzidine as the chromogenic peroxidase substrate, and slides were
counterstained with hematoxylin. Negative control samples included replacement
of the primary antibody with nonimmune IgG1 (Dako, Carpinteria, CA).
GSK-3β positive expression was defined as positive staining of
>50% of cancer cells throughout the neuroblastoma tumor
section.
TUNEL staining was performed according to manufacturer’s
protocol (ApopTag Detection Kit, EMD Millipore). The percentage of apoptosis was
calculated as the number of TUNEL-positive cells and bodies per 1000 cells
counted in three randomly selected microscopic fields in each xenograft tumor
sample.
neuroblastoma tissue microarray assay (TMA) and xenograft tumors. Paraffin
sections were deparaffinized, and antigen retrieval was carried out in citric
buffer in microwave for 10 min. The sections were incubated in 1%
hydrogen peroxidase for 10 minutes to quench endogenous tissue peroxidase.
Tissue sections were then incubated with the anti-GSK-3β antibody (Cell
Signaling, Danvers, MA) overnight at +4°C. The slides were stained using
a standard EnVision+ System-HRP kit (Dako, Carpinteria, CA) according to the
manufacture’s protocol. Immunohistochemical reactions were developed
with diaminobenzidine as the chromogenic peroxidase substrate, and slides were
counterstained with hematoxylin. Negative control samples included replacement
of the primary antibody with nonimmune IgG1 (Dako, Carpinteria, CA).
GSK-3β positive expression was defined as positive staining of
>50% of cancer cells throughout the neuroblastoma tumor
section.
TUNEL staining was performed according to manufacturer’s
protocol (ApopTag Detection Kit, EMD Millipore). The percentage of apoptosis was
calculated as the number of TUNEL-positive cells and bodies per 1000 cells
counted in three randomly selected microscopic fields in each xenograft tumor
sample.