Envision flex hrp polymer

Histopathology and PrP immunohistochemistry were performed as previously described [28 (link)]. Briefly, deparaffinized and rehydrated sections were immersed in Tris-buffered saline-Tween 20 (TBS-T) and endogenous peroxidase blocked by Envision Flex Peroxidase Blocking Reagent (Dako) for 10 minutes. Sections were washed, immersed in 1.5 mmol/L hydrochloric acid, microwaved for 15 minutes and incubated with 3F4 (1:1000) for 1 hour. After washing and incubation with Envision Flex/HRP polymer for 30 minutes (Dako), sections were treated with Envision Flex DAB (Dako) to show the immunostaining.

For the USA cases, the formalin-fixed brain was treated as previously described [18 (link)]. Tissue was deparaffinized, rehydrated, and immersed in Tween 20-Tris buffered saline. The endogenous peroxidase was blocked by the Envision Flex Peroxidase Blocking Reagent (Dako North America Inc., Carpinteria, CA, USA) for 10 min, which was followed by several washes. Sections were immersed in hydrochloric acid (1.5 mmol/L), microwaved for 15 min, and incubated with purified 3F4 Ab (1:1000) for 1 hour. Sections were washed and incubated with Envision Flex/HRP polymer for 30 min (Dako). The Envision Flex DAB (Dako) was used to show the immunostaining. Brain tissue blocks from the only UK-autopsied case were from 1985 and not suitable for modern immunocytochemistry.

To determine PD-L1 expression in HCC resection samples, serial 4–5 μm sections were stained with hematoxylin and eosin for histopathological assessment and were used for IHC. The diagnosis of HCC and the histologic subtype were confirmed independently by two board-certified pathologists (LMT and CI). Histologic grading and classification of HCCs were performed according to the World Health Organization (WHO) guidelines and the Edmondson & Steiner grading system (22 ). IHC was carried out using an Autostainer link 48 (Dako) with primary monoclonal antibodies recognizing PD-L1 (MKP-1A-73-10; PharmDx), CD8 (C8/144B; Dako), CD31 (JC70A; Dako), CD68 (PG-M1; Dako), FoxP3 (236A/E7; Abcam), and pan cytokeratin (AE1/AE3 Ventana). Envision FLEX HRP polymer and DAB⁺ (Dako) secondary reagents were used. For double labeling, the LabVision™ Multivision Polymer Detection System (ThermoScientific) was used according to the manufacturer's directions.