Antigen retrieval solution

The cancer tissue samples were fixed with formalin and embedded with paraffin. The expression of CD68 in TAMs was examined using immunohistochemistry on 5 μm thick whole mount tissue sections, and a Leica Bond MAX auto-stainer was used. The slides were dewaxed and antigenicity was retrieved using the Leica antigen retrieval solution, then the slides were incubated with the mouse monoclonal anti-human CD68 antibody (clone KP1, ZSGB-Bio) for 30 min. The staining was visualized using a diaminobenzidine system after secondary antibody incubation. The number of membranous CD68 positive cells in the whole tumor tissue sample was calculated and at least five randomly selected high power fields (400×) were examined. Original Hematoxylin-Eosin staining slices from the pathology archive were also reanalyzed to assess the presence and degree of TAMs.

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.