Ultravision hrp dab system

Human kidney normal-tumor paired tissue arrays KD1503 and KD1504 representing tumors in duplicates from 98 kidney clear cell carcinoma and their matched normal tissues were obtained from US Biomax, Rockville, MD, USA. Immunostaining of tissue arrays was performed as described [59 ]. Sections were dewaxed in xylene and rehydrated; antigen retrieval was performed by boiling the slides in 10 mm citrate buffer pH 6.0 for 20 min after. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in methanol for 15 min. After four washes of Tris buffer saline (20 mm Tris, 200 mm NaCl, pH 7.6), the sections were pre-incubated with Ultra V Block solution (Thermo scientific) for 10 min and incubated for 2 h with USPx antibody. Immunohistochemistry was performed using the streptavidin–biotin peroxidase complex method according to the manufacturer’s instructions (UltraVision HRP DAB system, Thermo scientific) using DAB as the chromogen. Individual tissue spots were examined by an experienced pathologist to confirm the tissue spot identity and visually assigned a score of 0 (no staining), 1 (weak staining of <10% of tissue), 2 (weak staining of 10–25% of tissue), 3 (weak to moderate staining of up to 50% tissue), 4 (moderate to strong staining of 50–75% of tissue) and 5 (moderate to strong staining of >75% of tissue).

Liver and kidney cancer arrays presenting tumors and adjacent normal tissue biopsy samples were obtained from US Biomax (Rockville, MD, USA; HLiv-HCC180Sur-02, HLiv-HCC180Sur-03 and HKid-CRC180Sur-01). Additionally, 72 archival patient samples from the pathology department, Rigshospitalet Copenhagen were examined. Ethical approval was obtained from the Danish National Committee on Biomedical Research Ethics. Immunostaining was performed using rabbit anti-ALDH7A1 (Sigma: HPA023296) [31 (link)] and the streptavidin–biotin peroxidase complex method according to the manufacturer’s instructions (UltraVision HRP DAB system, Thermo). Sections were examined by an experienced pathologist to confirm the tissue identity and assigned a score: 0 (no staining), 1 (weak staining up to 10% of tissue), 2 (weak staining 10–25% of tissue), 3 (weak to moderate staining ≥50% of tissue), 4 (moderate to strong staining of 50–75% of tissue) and 5 (moderate to strong staining > 75% of tissue). The score for each tumor was calculated by subtracting the score of the normal tissue from that of that tumor.