Optiview dab immunohistochemistry detection kit

All the buffy coat samples that were studied histopathologically were immunohistochemically stained and placed in a VENTANA^® Benchmark ULTRA/LT automatic immunohistochemistry processor (Ventana Medical Systems, Oro Valley, AZ, USA). The standardized protocol for SARS-CoV-2 detection was used: a recovery solution of pH 9 for 40 min at 100 °C and the Optiview^® DAB Immunohistochemistry Detection Kit (VENTANA^®). The primary anti-SARS-CoV-2 NP antibody, clone 6F10 (BioVision Incorporated^®, Milpitas, CA, USA), and the SARS-CoV-2 spike protein S2 antibody, MA5-35946 (Invitrogen^®, Carlsbad, CA, USA), were reconstituted with 100 mL of distilled water at a dilution of 1:1000 and incubated for 32 min at 36 °C. Finally, the histological slides were developed with diaminobenzidine, contrasted with Mayer’s hematoxylin, dehydrated with alcohols at increasing concentrations, rinsed with xylol, and finally examined under an Olympus BX41 light microscope with direct increases ranging between 3.5× and 60×. SARS-CoV-2 positivity was considered when cytoplasmic granular labeling was obtained in cells from the buffy coat samples. Positive external controls for each antibody were used. RT-PCR SARS-CoV-2 assay results were unknown at the time that the immunohistochemical studies were evaluated.

We assessed the protein expression levels of ER, PR, and HER2 in the BC tissue samples. The tissue sections were stained using the Ventana BenchMark ULTRA automatic immunohistochemical staining platform (Ventana Medical Systems Inc., Tucson, AZ, USA) and observed under a microscope (Olympus BX41). Rabbit monoclonal primary antibodies against ER (SP1 Roche), PR (1E2 Roche), HER2/NEU (Clone 4B5 Roche), and PD-L1 (SP142 Roche) were used, and the OptiView DAB immunohistochemistry Detection Kit and OptiView Amplification Kit (Ventana Medical Systems Inc.) were used for subsequent analysis. HER2 staining was scored according to the HER2 Testing Guidelines for Breast Cancer (2019 edition) (13 ). ER- and PR-positive staining was defined according to the ASCO/CAP guidelines (14 (link)).
In the HE slides, TILs were defined as a continuous parameter by two experienced pathologists. TILs on the boundaries of the cancer were included, while those in the tumor bed were excluded, and they were scored based on the area occupied over the entire region. The final percentage of TILs was calculated as the average of the specimens and was not restricted to hotspots. All evaluations were performed according to the criteria recommended by the International TILs Working Group (2014) (15 (link)). TILs were assessed as a continuous parameter, and reported scores were rounded up to the nearest 10%.