Polymer refine detection system

The staining procedure was performed using a Leica Bond III Stainer (Leica,). Slides were subjected exposed to 10 mM sodium citrate buffer, pH 6.0 for 20 min at 37°C. Slides were incubated with the appropriate primary antibody for 15 min, followed by Polymer Refine Detection System (Leica) processing, including hydrogen peroxidase block, secondary antibody polymer, 3,3’ diaminobenzidine and hematoxylin stain. Specimens were then rinsed for 5 min in tap water. Slides were dehydrated in increasing concentrations of ethyl alcohol and xylene prior to permanent coverslipping in xylene-based media. Primary antibodies were as follows: mouse anti-Hif1α (1:400, Novus Biological), mouse anti-HIF2α (Novus Biologicals, NB100-132, rabbit polyclonal, 1:700) mouse anti-H3K9me2 (1:750, Abcam), rabbit anti-H3K27me2 (ab24684, Abcam, 1:500), rabbit anti-5hmC (39769, Active Motif, 1:4000), and mouse anti-SDHB (ab14714, Abcam, 1:1000).

The immunostaining (IHC) experiment was performed at the Mayo Pathology Research Core and followed our previously published protocol^{31 (link)} with minor modifications. Briefly, the staining procedure used Leica Bond RX Stainer (Leica, Buffalo, IL). The frozen slides were pulled from −80° and placed in a desiccator to dry overnight. The slides were fixed in 4% paraformaldehyde for 10 min and then rinsed in PBS. The slides were loaded wet onto the stainer and the epitope retrieved on-line using Epitope Retrieval 2 (EDTA; Leica, Buffalo, IL) for 10 min. The primary antibody TopoIIA (Clone 3F6, Leica Microsystems) was diluted to 1:200 in Bond Diluent (Leica) and incubated for 15 min.
The detection system used was Polymer Refine Detection System (Leica, Buffalo, IL). This system includes the hydrogen peroxidase block, secondary antibody polymer, DAB, and hematoxylin. Once completed, slides were removed from the stainer and rinsed for 5 min in tap water. Slides were dehydrated in increasing concentrations of ethyl alcohol and xylene prior to permanent coverslipping in xylene-based media.

Rat brains were dissected and fixed in 10% neutral buffered formalin for 24 hours and transferred to 70% ethanol for processing into paraffin blocks. The antibodies used are shown in Supplementary Information and stained sections were imaged using Leica’s Polymer Refine Detection System on the Bond-III platform.

Paraffin-embedded samples of human lung cancer tissues were cut into 4-μm sections, mounted on coated glass slides, and incubated with anti-AXL antibody (1:100, #8661, Cell Signaling Technology), and anti-CDCP1 antibody (1:100, #4115, Cell Signaling Technology). Briefly, immunostaining with each antibody was performed on the same fully automated Bond-III system using onboard heat-induced antigen retrieval with epitope retrieval solution 2 (pH 9.0) for 30 min, and incubated with each antibody for 30 min. This automated system uses a Refine polymer detection system (Leica Microsystems, Newcastle, UK) with HRP (horseradish peroxidase)-polymer as secondary antibody and 3,3′ diaminobenzidine (DAB) as the chromogen. The slides were visualized using DAB.