Ultra cc1

Slides were pretreated with ultraCC1 (proprietary, Ventana Medical Systems Inc., Tucson, AZ) for 64 minutes and stained using a rabbit monoclonal primary antibody (Abcam, Cambridge, MA, ab68478, 1:10). UltraView DAB detection kit (proprietary, indirect, biotin free system, Ventana Medical Systems Inc., Tucson, AZ) was used for primary antibody detection.

Tissue samples were obtained from formalin-fixed and paraffin embedded blocks from surgical samples. 2 μm thick sections were used for routine Hematoxylin and Eosin (H&E) staining and immunohistochemistry using the automatic stainer BenchMark ULTRA IHC/ISH System (Ventana). Diagnosis of cortical cell carcinoma was revised according to the most recent WHO criteria (30 ). The clinical characteristics of the patient are reported in Supplemental Table 1. The following primary antibodies were used: anti-PgR clone 1E2, anti-ER clone SP1. All the primary antibodies were from “ready to use” kits from Ventana. Antigen retrieval was performed by incubation for 64 min for PgR and ER at 95°C in Ultra Cell Conditioning Solution (Ultra CC1, Ventana). Signal was revealed using the ultraView Universal DAB Detection kit (Ventana) followed by diaminobenzydine as chromogen and Hematoxylin for nuclear counterstain. Digital images were acquired by an Olympus XC50 camera mounted on a BX51 microscope (Olympus, Tokyo, Japan) using CellF Imaging software (Soft Imaging System GmbH, Münster, Germany). Expression of PR and ER was semi-quantitatively scored on representative tumor areas based on both percentage [score ranges: 0 (0–5%), 1 (6–29%), 2 (30–69%), 3 (≥70%)] and intensity (score ranges: 0, no expression; 1, weak; 2, moderate; 3, high) of immunoreactive (IR) neoplastic cells.

Three-micrometer-thick sections were cut from paraffin blocks containing tumor tissue and surrounding normal liver tissue using a microtome. After deparaffinization and rehydration, the samples were pre-treated with Cell Conditioning Solution (Ultra CC1; Ventana, Oro Valley, USA) for 32–48 min (SAA, LFABP, and GS: 32 min; beta-catenin: 40 min; and CRP: 48 min). Immunohistochemical stainings were carried out using an automated slide stainer (BenchMark Ultra system; Roche, Basel, Switzerland) using the following dilutions: SAA—1:200; LFABP—1:1000; and GS and beta-catenin—ready-to-use. The list of antibodies used in the study is provided in Supplementary Table 1.

All samples were subjected to H&E staining to detect signs of cytopathic damage (i.e., inclusion bodies). IHC staining for HCMV was performed on all formalin-fixed paraffin-embedded (FFPE) samples, sectioned at 4 μm on polarized slides. HCMV IHC was performed using a Benchmark ULTRA staining module (Ventana Medical Systems, Roche Diagnostics, MB, Italy) equipped with a fully automated protocol. Briefly, slides—previously baked overnight at 37 °C—were deparaffinized and rehydrated. Antigen retrieval was achieved using UltraCC1 (Ventana Medical Systems) for 36 min. The prediluted HCMV antibody (anti-HCMV blend cocktail 8B1.2, 1G5.2 & 2D4.2 mouse monoclonal primary antibody; catalogue No. 760-4703; Ventana Medical Systems) was incubated for 32 min at RT. Signal amplification was performed with the Amplification kit (Ventana Medical Systems), and detection was carried out with UltraView Universal DAB Detection kit (Ventana Medical Systems). To enhance visualization, counterstaining was performed with hematoxylin for 8 min, followed by 4-min incubation with Bluing Reagent (Ventana Medical Systems). Upon completion of the automated staining, the slides were subjected to dehydration and coverslipping through an HE600 platform (Ventana Medical Systems; Roche Diagnostics).

Slides were pretreated with ultraCC1 (proprietary, Ventana Medical Systems Inc., Tucson, AZ) for 36 minutes and stained using a rabbit polyclonal primary antibody (Abcam, Cambridge, MA, ab11011, 1:1000). UltraView DAB detection kit (proprietary, indirect, biotin free system, Ventana Medical Systems Inc., Tucson, AZ) was used for primary antibody detection.
β-catenin (VentanaMed Systems Inc, Tucson, AZ) was used as a prediluted antibody after antigen retrieval.
All slides were counterstained with hematoxylin and routinely dehydrated, cleared, and cover- slipped in resinous mounting media. Staining intensity for each of the abovementioned markers was evaluated in each of four different HBL tumor components, fetal (n = 51), embryonal (n = 29), and SCU (n = 10). The less common mesenchymal cell type (n = 4) was not analyzed. The distribution of staining was also noted (membranous versus cytoplasmic versus nuclear) for all stains. Stain controls, positive and negative, were used for all stains and runs. Between-group comparisons were performed with chi-square test.