Dp2 bsw image acquisition software system

IHC was carried out according to the manufacturer's protocol. Briefly, formalin‐fixed and paraffin‐embedded tissue sections were deparaffinized in xylene and hydrated through descending concentrations of ethanol before being placed in blocking solution to inhibit endogenous peroxidase activity. The slides were incubated with primary antibodies at 4°C overnight. A horseradish peroxidase‐conjugated rabbit or mouse secondary antibody was added for 60 min at room temperature, followed by 3, 3′‐diaminobenzidine (DAB) development (DAB Substrate Chromogen System, Dako) and hematoxylin and eosin (H&E) as per standard staining protocol. Slides were fixed and images obtained with the Olympus IX71 inverted microscope using the DP2‐BSW Olympus image acquisition software system. The results were confirmed by two experienced pathologists who were blinded to the clinicopathologic data of the patients. The staining results were calculated on the basis of the percentage of tumor cell nuclei stained (0, no staining; 1, ≦10%; 2, 10–50%, and 3, >50%) and the staining intensity (0, negative; 1, weak; 2, moderate; and 3, strong). An overall score of 1–5 designated low expression, and an overall score of 6–9 designated high expression for both CSN6‐ and EMT‐related genes in PTC tissues.

IHC staining was carried out according to the manufacturer’s instructions. Briefly, formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol (100, 95, 80 and 75%). The slices were then soaked in 10% BSA to inhibit endogenous peroxidase activity and incubated with anti-PD-1 or anti-LAG3 rabbit polyclonal antibody (1:100; Cat: 86163&15372, Cell Signaling Tech, Boston, MA USA) at 4°C overnight. A horseradish peroxidase (HRP)-conjugated rabbit secondary antibody (1:400; Cat: 8114, Cell Signaling Tech) was added for 60 min at room temperature; then, 3,3’-diaminobenzidine development (DAB Substrate Chromogen System; Dako) and hematoxylin staining were performed according to standard protocols. Slides were fixed and images were obtained with an Olympus IX71 inverted microscope and a DP2-BSW Olympus image acquisition software system.

Formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated with ethanol. Blocking of endogenous peroxidase activity was conducted using 3% hydrogen peroxide. After heat-induced antigen retrieval (0.01 mol/L citrate, pH 6.0), 5% bovine serum albumin was used to block nonspecific protein-protein interactions. Sections were then incubated overnight with primary antibodies against IL-10 (20850-1-AP, Proteintech), HLA class I ABC (ab70328, Abcam) and PD-L1 (13684T, Cell Signaling Technology). Secondary antibody staining and antigen detection were performed using a horseradish peroxidase–conjugated rabbit or mouse immunohistochemistry (IHC) kit (KIHC-1, Proteintech). Sections were counterstained with hematoxylin. Images were obtained through an Olympus IX71 inverted microscope with a DP2-BSW Olympus image acquisition software system (Olympus, Japan).

IHC was carried out according to the manufacturer's protocol. Briefly, formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and hydrated with decreasing concentrations of ethanol before being placed in a blocking solution to inhibit endogenous peroxidase activity. The slides were incubated with primary antibodies at 4°C overnight. A horseradish peroxidase-conjugated rabbit or mouse secondary antibody was added for 60 min at room temperature; then, 3, 3′-diaminobenzidine (DAB) development (DAB Substrate Chromogen System, Dako) and hematoxylin and eosin (H&E) staining were performed according to standard protocols. Slides were fixed and images were obtained through an Olympus IX71 inverted microscope with a DP2-BSW Olympus image acquisition software system. The results were confirmed by two experienced pathologists who were blinded to the clinicopathologic data of the patients. The staining results were calculated on the basis of the percentage of tumor cell nuclei stained (0, no staining; 1, ≤10%; 2, 10%-50%; and 3, > 50%) and the staining intensity (0, negative; 1, weak; 2, moderate; and 3, strong). These scores were multiplied, such that an overall score of 1-5 designated low expression, and an overall score of 6-9 designated high expression for both YAP1 and the proliferation-related proteins in the 50 PTC tissue samples [17 (link)].