K8005

Specimens were pretreated using a 3-in-1 procedure [deparaffinization, rehydration, and target retrieval in the PT Link (code PT100/PT101/PT200)] using a low pH (code K8005; Dako North America Inc.). Subsequently, they were stained on the Autostainer Link 48 (code AS480), an automated IHC testing platform with a staining protocol validated for PD-L1 IHC 22C3 pharmDx, using the PD-L1 IHC 22C3 pharmDx reagents and protocol (package insert22 ). The stained specimens were counterstained with hematoxylin (code K8008; Dako North America Inc.) and coverslipped.

Subcutaneous adipose tissue samples were immediately immersed in 10% neutral buffered formalin fixative after sampling for 24 hours before storage in PBS at 4°C. After paraffin embedding, 3-μm thickness paraffin sections were dewaxed (successive toluene and descending alcohol baths) and stained with hematoxylin and eosin. Labeling of 3-μm sections of paraffin-embedded adipose tissues samples was performed after antigen retrieval (Ptlink low pH, reference K8005, Dako) for 30 minutes, using anti–human-specific Ku-80 antibody (2753S Cell Signaling Technology, rabbit monoclonal antibody, dilution 1:150) and incubated for 50 minutes at room temperature. Staining was carried out with Dakostainer automated system using a biotinylated anti-rabbit antibody (reference ABK125 Microm) for 25 minutes at room temperature, followed by HRP (1:150, 25 minutes room temperature) and DAB as a chromogen. The stained slides were imaged by light microscopy on a Nikon Eclipse Ci-L microscope with a DS138 Fi3 Camera and NIS Elements D software.

The FFPE tissues, including TMAs, were sectioned at thickness of 6 μm and placed on slides. These specimens were heated at 65°C for 2 h, deparaffinized in xylene for 10 min, and dehydrated with 100% ethanol. The specimens were treated for 30 min with pretreatment solution (K8005; Dako) at 95°C to improve the accessibility of DNA, and were proteolyzed in 50 mM Tris–HCl (pH 7.6) with protease K at 37°C for 25 min, which was deactivated with 50 mM MgCl₂ for 10 min at room temperature. The slides were then postfixed in 10% neutral buffered formalin for 2 min, and dehydrated in a graded ethanol series.
A mixture of FISH probes was applied onto the specimens, overlayed, and sealed with cover slips. The slides were denatured at 75°C for 5 min, and hybridized at 37°C for 48 h. After removing the cover slips, the slides were washed in 2× SSC (pH 7.0) with 0.3% NP‐40 at 72°C for 2 min and were rinsed in 2× SSC. Slides were mounted with a medium containing DAPI. The DNA FISH probes for the centromeres of chromosome 6 and 16 were purchased from Vysis (CEP 6 [D6Z1] SpectrumOrange Probe and CEP 16 [D16Z3] SpectrumGreen Probe).
Cultured cells including RPE1, HeLa, and U251 cells were fixed with 3:1 methanol : acetic acid, dropped onto glass slides, and dried. The remaining steps of DNA FISH were followed by processes as described above.