Ar9661

IF was performed on 4-µm FFPE sections that had previously been kept at 60 °C for 2 h. FFPE sections for AHCY (10757-2-AP, Proteintech) IF staining were loaded onto a Leica Bond Rx autostainer. FFPE sections underwent on-board dewaxing (AR9222, Leica) and antigen retrieval using ER1 solution (AR9661, Leica) for 40 min at 95 °C. Sections were rinsed with Leica wash buffer (AR9590, Leica) before a 10% normal goat serum (X090710, Agilent) solution was applied for 30 min. Sections were rinsed with wash buffer before AHCY antibody was applied at a 1:500 dilution. Sections were rinsed with wash buffer and anti-rabbit IgG 647 diluted at a 1:250 ratio applied for 30 min before rinsing with wash buffer. DAPI was applied to the sections before rinsing with wash buffer and then the sections were coverslipped using pro-long gold (P10144, Thermo Fisher). Images were captured using the Keyence BZ-X810 microscope.

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.