Ultraview universal dab kit

Immunohistochemical staining was used to detect the expression patterns of GPC3, CK19, vimentin, p53, AFP, EGFR, RRM1, BRCA1, VEGF, TS, Ki 67, ARG and GS in HCC tissues. The staining procedure was performed according to the instructions of the Roche benchmark XT Ventana (Roche Diagnostics, USA) automatic staining instrument, and the paraffin sections were detected by automatic immunohistochemistry. The slices were dewaxed, pretreated with cleaning buffer for 30 min, and then washed with reaction buffer. Subsequently, the following primary antibodies were added: GPC3, CK19, vimentin, mutant p53, AFP, EGFR, VEGF (all sourced from Santa Cruz Biotechnology, USA), RRM1, and BRCA1 (both sourced from Abcam, UK), After incubation for 30 min, the cells were stained with ultraView Universal DAB Kit (Roche Diagnostics, GER).
Positive criteria: All immunohistochemical staining results were judged independently by two senior pathologists, and unanimous interpretations are made in the event of a disagreement. The agreement between the two experts on the positive results of immunohistochemical staining was 97.15% (2526/2600). Furthermore, 74 immunohistochemical stained sections with inconsistent judgments were agreed upon after consultation.

Formalin-fixed and paraffin-embedded (FFPE) archival tumour blocks were analysed by immunohistochemistry by collecting 4 μm sections on Superfrost charged slides (ThermoScientific). After drying overnight at 37 °C, samples were processed using a Ventana Benchmark immunohistochemistry platform (Roche) with antibodies against p53 (Agilent cat#7001, RRID: AB_2206626, 1:50), CK7 (Agilent cat#7018, RRID: AB_2134589, 1:250), PAX8 (Roche cat#760–4618, 1:100). Heat induced epitope retrieval was performed using CC1 (Roche), incubating samples at 95 °C for 36, 52, and 40 min for p53, CK7 and PAX8, respectively. Antibodies were incubated at 37 °C for 32, 40 and 32 min for p53, CK7 and PAX8, respectively. p53 and CK7 were detected using Ultraview universal DAB kit (Roche), while PAX8 was detected using Optiview universal DAB kit (Roche), all as per manufacturer’s instructions. Sections were counterstained using Haematoxylin II (Roche) for 12 min and bluing reagent (Roche) for 8 min. Slides were imaged using the EVOS FL Auto 2 Imaging System (Invitrogen), using a × 10 or × 40 objective lens under bright field, and processed using Adobe Photoshop.