Immunohistochemical Analysis of GLS in RCC

RCC tumors were purchased from Indivumed (Hamburg, Germany). Immunohistochemical staining was performed at Covance Laboratories Inc. (Greenfield, USA), as previously described [30 (link)]. Tumors were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into 4 μm sections, which were then dried, deparaffinized, and rehydrated. Heat-induced antigen retrieval was performed for 10 minutes (Antigen Retrieval Solution, Leica, AR9661). Endogenous peroxidase activity was quenched with peroxidase block. Sections were incubated with primary antibody (rabbit anti-human GLS, Abcam, ab156876, 1:200 dilution) for 15 minutes at room temperature. Immunohistochemical reactions were visualized using Dako Rabbit Envision+HRP with DAB+ for use with rabbit primary antibodies (K4011, Dako). Sections were counterstained with hematoxylin.