Vectamount permanent mounting media

Tumour biopsy sections (4–5 μm) were de-paraffinised and rehydrated, with antigen retrieval in citrate buffer (pH 6) and unspecific binding sites blocked with 1.5 % horse serum in PBS. The sections were then stained with mAb p16^INKA4a (clone: JC8, dilution 1:100, Santa Cruz Biotech, Santa Cruz, CA, USA) at +8 °C overnight, before incubation for 45 min. with biotinylated anti-mouse antibody (dilution 1:200, Vector Laboratories, Burlingame, CA, USA). Alternatively, the slides were stained with the CINtec® p16 Histology (805–4713), Ventana Medical Systems Inc., Arizona, USA by following the same protocol with the exception of incubation with the antibody for 1 h at room temperature. For antigen detection, the avidin–biotin–peroxidase complex (ABC) kit (Vectastain, Vector Laboratories, Burlingame, CA, USA) was used. Slides developed in chromogen 3’-diaminobenzydine (DAB) (Vector Laboratories, Burlingame, CA, USA) and counterstained with haematoxylin were then washed and dehydrated, and the cover mounted using VectaMount permanent mounting media (Vector Laboratories, Burlingame, CA, USA). P16 staining was regarded as positive if >70 % of the tumour cells were strongly p16-positive [28 (link), 29 ].

FFPE sections underwent hydration, antigen retrieval, avidin–biotin blocking, and incubation with blocking buffer and primary and secondary Abs as described above. Endogenous peroxidase was quenched with a 3% solution of hydrogen peroxidase for 30 min prior to antigen retrieval. For slides stained with rabbit pAb to the ionized calcium‐binding adapter molecule 1 (Iba1; Wako, Richmond, VA) diluted 1:1000 in blocking buffer, the secondary was a biotinylated anti‐rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The signal on these slides was amplified with the Avidin–Biotin–HRP complex (ABC; Vector Laboratories) before being developed with the 3,3′‐diaminobenzidine tetrachloride (DAB) substrate (Vector Laboratories) as the chromogen. For slides stained with rabbit pAb to TREX1 (diluted 1:200 in blocking buffer), the secondary was a peroxidase‐conjugated anti‐rabbit IgG (Jackson ImmunoResearch Laboratories). These slides were developed with DAB. Coverslips were mounted with VectaMount Permanent Mounting Media (Vector Laboratories).

Slides were baked 1 h at 65 °C, before deparaffinization and hydration in xylene and graded ethanol to distilled water. Endogenous peroxidase was blocked for 5 min in 1.5% H₂O₂ in methanol, antigen retrieval step was performed by boiling slides for 20 min in “Antigen unmasking solution” (Vector Laboratories) and cooling down 1 h at RT. Slides are quickly washed in PBS for 5 min, blocked in PBS-2% BSA-5% FBS 1 h at RT, and incubated overnight at 4 °C in a humid chamber with primary antibody. After washing three times for 5 min in PBS, slides were incubated with biotinylated secondary antibody (Vector Laboratories) for 30 min. Slides were washed in PBS three times for 5 min, before incubating with the ABC substrate for 30 min at RT. After washing again with PBS, DAB was prepared according to the manufacturer instruction (Vector Laboratories), and the staining reaction monitored from 1 to 5 min. Slides were stained with H/E following standard methods, dehydration steps from 90% ethanol solution to xylene is performed, and slides were mounted in VectaMount permanent Mounting Media (Vector Laboratories).