Ultravision quanto detection system hrp dab

Post‐fixed tissues samples were stained with: (i) anti‐CD3, anti‐CD20, and anti‐CD11c to assess the nature of infiltrating cells. Detection of signal was performed with UltraVision Quanto Detection System HRP DAB (Thermo Scientific^™); (ii) anti‐ARSA antibody recognizing an epitope conserved in both human and Macaca fascicularis by using Ventana autostainer. Samples included cortical and subcortical white and gray matter regions of the left and right hemispheres of slices 3–6. All specimens were examined in blind. ARSA‐positive signal was quantified, and a score was assigned based on the following factors: pattern of distribution of stained cells, approximate intracellular granulation size and number (low versus high quantity of granules), and the level of the staining in the surrounding extracellular matrix (ECM). The sum of the partial scores gave the final grade for each specimen. For each NHP, the mean score was calculated and data were expressed as fold increase to the score assigned to P2 NHP (control).

Primary tumor sections of 4-μm thickness were deparaffinized and pre-treated for antigen retrieval by autoclave heating (121 °C) in 10 mM sodium citrate buffer (pH 6.0) for 10 min. These sections were blocked for endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 min and then washed in phosphate-buffered saline (PBS). Thereafter, the sections were immersed in UltraVision Protein Block (Thermo Fisher Scientific, Fremont, LA, USA) for 10 min, covered with a primary rabbit monoclonal antibody specific for p16 (clone: EP1215Y, Epitomics, Abcam Company, Burlingame, CA, USA) and incubated for one hour at room temperature. Immunoreactions were performed using UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, Fremont, LA, USA). Immunohistochemical evaluation of p16 in OPSCC specimens was based on the intensity and extent of nuclear and cytoplasmic reactivity. Positive p16 expression was defined as strong and diffuse nuclear and cytoplasmic staining in 70% or more of the tumor cells^{6 (link)}. Two independent pathologists (Y-L.C. and C-T.W.) were involved in the assessment of tumor p16 expression.

Tissue sections were collected and fixed in 10% formalin. 5 μM slides were used for immunohistochemistry analysis. The “UltraVision Quanto Detection System HRP DAB” IHC kit (TL-125-QDH, Thermo Fisher Scientific) was used for the tyramide signal amplification according to the manufacturer’s protocol. Primary antibodies used in this assay are as follows: anti-CD20 (M0755, Dako), anti-MLH1 (MAB-0789, MXB Biotechnologies), anti-MSH2 (MAB-0836, MXB Biotechnologies), anti-PMS2 (GT215902, Gene Tech) and anti-MSH6 (MAB-0831, MXB Biotechnologies). Images were taken and quantitative image analysis was performed using ImagePro software. For comparison for CD20 between two groups, statistical evaluation was done by two-tailed Student’s t-test, error bars show standard error of the mean (SEM).

Standard immunohistochemical procedure was used for staining FFPE sections with antibodies against stromal cell markers. We used the following antibodies: mouse anti-CD163 (Clone 10D6; BIOCARE, USA, 1 : 100 dilution), rabbit anti-CD206 (HPA004114; Sigma, USA, 1 : 2000 dilution), rabbit anti-iNOS (SAB5500152; Sigma, USA, 1 : 150 dilution), rabbit anti-FOXP3 (Clone D2W8E; Cell Signaling Technology, USA, 1 : 200 dilution), rabbit PU.1 (Clone 9G7; Cell Signaling Technology, USA, 1 : 200 dilution), rabbit anti-PD-L1 (E1L3N; Cell Signaling Technology, USA, 1 : 200 dilution), rabbit anti-CD68 (Clone GR021, 61-0184 Genemed, USA, 1 : 100 dilution), mouse anti-CD8 (Clone CD8/144B, 61-0124 Genemed, USA, 1 : 100 dilution), and rabbit anti-CD3 (61-0011 Genemed, USA, 1 : 100 dilution). We used UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, USA).

For IHC staining of PD-L1 expression in tumor tissue, 4-μm-thick sections from each formalin-fixed, paraffin-embedded tissue blocks were de-waxed with xylene and rehydrated in a graded series of ethanol. For PD-L1 (a rabbit PD-L1 polyclonal antibody which has been used in several previous studies) [6 (link), 16 (link), 17 (link), 19 (link), 27 (link), 28 (link), 29 (link)] Proteintech group Inc., Chicago, IL, USA. The sections were incubated with the PD-L1 antibody (1:250 dilution) for 1h, and antigen retrieval was performed by autoclaving for 9 min at 121°C in citrate buffer, pH 6.0. The UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, Fremont, CA, USA) was used according to the manufacturer's instructions. The sections were counterstained with hematoxylin and then mounted.
PD-L1 immunostaining results were classified into two groups according to the intensity and extent of staining: (1) negative, no staining or staining detected in < 5% of the cells; and (2) positive, when membranous staining was present in ≥ 5% of the cells and the staining intensity was moderate to strong. The 5% threshold was based on a previous phase 1 trial of anti-PD-1 agents and studies of other malignances [8 (link), 11 (link), 12 ]. Two independent pathologists (C-T. W. and Y-L. C.) assessed PD-L1 expression.

Immunohistochemical staining was utilized to observe SOD and GPx expression. Kidney tissue slices of 4 μm were deparaffinized and was added hydrogen peroxide at 37°C for 10 minutes to inhibit endogenous peroxide. Then, 10% normal sheep serum was given in Tris-buffered salt solution at 37°C for 30 minutes. Furthermore, incubated overnight at 4°C with anti-rat anti-SOD monoclonal (1:100; ab8376; Abcam, Cambridge, MA, USA) or anti-rat anti-GPx monoclonal (1:100; sc8008, Santa Cruz Biotechnology) antibodies. After that, We washed three times with PBS and incubated with a secondary antibody from the UltraVision Quanto Detection System HRP DAB (Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes at room temperature, and with 3.3′ diaminobenzidine (DAB) color reagent. Immunohistochemical expressions were observed by microscopy and semiquantified by Image-Pro Plus 6.0 software. The integrated optical density (IOD) of each photo was collected. Images were measured by immunoreactive area (IA) in μm² and IOD. The staining intensity (SI) for each image was calculated as SI = IOD/IA and the mean with standard deviation.