Envision flex visualization system

Immunohistochemistry for pan T-cell marker CD3 was performed on 4 µm-thick tissue sections of formalin-fixed paraffin-embedded blocks using an automated immunostaining system (GI100, Dako Omnis, Agilent Technologies, Santa Clara, CA, USA). Antigen retrieval was performed with high-pH EnVision FLEX Target Retrieval Solution (Agilent Technologies) for 30 min at 97 °C. Tissues sections were incubated with polyclonal rabbit anti-human CD3 antibody (1:100, Code No. A 0452, Agilent Technologies) for 20 min at room temperature, followed by visualization with EnVision FLEX visualization system (EnVision/HRP for 20 min and chromogen substrate for 5 min). The specimens were then counterstained with Hematoxylin for 3 min.

After formalin fixation and paraffin embedding, three to four haematoxylin and eosin-stained sections were routinely prepared from each prostate biopsy, as well as an additional unstained section for any additional immunohistochemical studies that may be required. Immunohistochemical analysis was performed using the automated staining platform DAKO Omnis (Dako/Agilent, Santa Clara, CA, USA) on 1–2-µm-thick sections from formalin-fixed paraffin embedded prostate biopsies. The following antibodies were used: p63 (clone DAK-p63, DAKO/Agilent, mouse monoclonal, ready to use), cytokeratin 5/6 (clone D5/16 B4, DAKO/Agilent, mouse monoclonal, ready to use), and high molecular weight cytokeratins (clone 34ßE12, DAKO/Agilent, mouse monoclonal, ready to use) in combination (double staining) with an antibody to Alpha-methyl acyl coenzyme-A racemase (AMACR) (clone 13H4) DAKO/Agilent, rabbit monoclonal, ready to use. The antibodies were configured as FLEX Ready-to-Use (Agilent) and used with the EnVision FLEX visualization system (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions for use.

IHC analysis was carried out in previously deparaffinized tissue sections. Rehydration and posterior antigen retrieval were performed using XS Tris Buffered Saline with Tween 20 and boiled for 20 min. Rabbit monoclonal primary PD-L1 antibody (Monoclonal Mouse Anti-Human PD-L1 Clone 22C3, Agilent Technologies, Santa Clara, California, US) was processed using 4 mm-thick FFPE tissue sections on a EnVision FLEX visualization system on Autostainer Link 48 (Agilent Technologies, Santa Clara, California, US) with standard antigen retrieval methods. The Signal Stain DAB substrate kit (#8959) was used according to the manufacturer´s instructions. Human placenta was used as positive control. PD-L1 protein expression was determined using Tumor Proportion Score (TPS), which is the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. Tumors with ≥1% of tumor cells stained either in membrane or cytoplasm will be considered positive for PD-L1. The grading system of PD-L1 expression was: 0 (negative), 1–49%, (weak to moderate expression), and >50 (strong expression).

The pathological diagnosis of pituitary adenomas (PitNETs) was performed by one pathologist at the Department of Pathology and Laboratory Diagnostics, Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland. Pathological evaluation for pituitary tumors (PitNETs) included routine immunohistochemical staining on GH, PRL, ACTH, β-TSH, β-FSH, β-LH, alpha subunit, and Ki-67. In selected cases, immunostaining was extended to the expression of Cam5.2, SSTR2A, SSTR5, p53, MGMT, Collagen IV and others, when appropriate.
The standard immunohistochemical evaluation was performed using 10% buffered formalin, in which tissue samples were embedded and stained with hematoxylin and eosin. Immunohistochemical staining was performed on paraffin-embedded sections according to the labeled EnVision Flex Visualization System (K8000, Dako/Agilent) with DAB (3,3′-diaminobenzidine). For the ACTH evaluation we used antibody from Thermo Fisher Scientific (concentration 1:500; cat. no.: M5-1452-P1) and Ki-67 from Dako company (MIB-1 clone; ready to use antibody) as well as Anti-Cytokeratin antibody from Ventana company (Cam 5.2, cat. no.: 790-4555).

The PD-L1 IHC 22C3 pharmDx IHC assay (Agilent Technologies/Dako, Carpinteria, California, USA) was performed in all cases. All tissue samples underwent fixation in 10% neutral buffered formalin for 6-24 hours. Four-μm thick formalin-fixed, paraffin-embedded tissue sections were dried at 60°C for 30 minutes. PD-L1 IHC 22C3 pharmDx IHC assays were performed using an EnVision FLEX visualization system (Agilent, Santa Clara, USA) and an Autostainer Link 48 system (Dako). In accordance with the instructions of the manufacturer, positive and negative controls were used. All cases were stained within 6 months of sectioning.
The Tumor Proportion Score (TPS) is defined as the percentage of viable tumor cells with partial or complete PD-L1 membranous staining at any intensity (≥ 1+) relative to all viable tumor cells in the sample (3 ). The IHC results of 60 cases were evaluated by four pathologists and TPS was assigned by each. TPS was categorized into three expression levels: no/low expression (<1%), intermediate expression (1–49%) and high expression (≥50%) (Figure 1).

Rictor IHC was performed on 4–µm–thick sections of the same blocks. After deparaffinization and blocking endogenous peroxidases, antigen retrieval was performed for 30 min (10 mM citrate, pH 6.0) in a pressure cooker. Slides were then incubated with an anti-Rictor (1:1000; #A500-002A; Bethyl Laboratories, Montgomery, TX, United States) primary antibody. Vectastain Universal Elite ABC HRP Kit (Vector Laboratories, Newark, CA, United States) secondary detection system and DAB (Agilent, Santa Clara, CA, United States) chromogen were used to visualize the reactions. The sections were counterstained with hematoxylin.
PD-L1 IHC 22C3 PharmDx reactions were performed on an automated immunostainer (Leica BOND-III; Leica) using the EnVision FLEX visualization system (Agilent) according to the manufacturer’s protocols.

All samples were sectioned and stained on the same occasion for comparable analysis. Paraffin-embedded sections were placed in a PT Link Pre-treatment Module (Agilent) 97 °C for 20 minutes for deparaffinization, followed by incubation with Target Retrieval Solution, Citrate pH 6 (S236984–2, Agilent) for 30 minutes. Immunohistochemistry was performed in an Autostainer Link 48 (Agilent) using an EnVision FLEX visualization system (Agilent) and counterstained with hematoxylin-eosin. Tissue sections were incubated for 30 minutes at room temperature with primary antibody, a polyclonal rabbit anti-human VAP-1 antibody (1:100, PA5–81910 Thermo Fischer Scientific). Negative control sections were prepared by performing immunostaining procedures without adding primary antibodies. Stained sections were scanned by a digital slide scanner (NanoZoomer S60, Hamamatsu) using the same exposure times. Digitalized sections were examined by NDP.view2 (Hamamatsu), a whole slide viewing software. The same magnification (10 x objective) was used for all the images. Three representative areas per section/patient were exported into three images (size, 23 MP, 6400 × 3616 pixels, type: RGB, format: TIFF). RGB image allowed the range of 255 intensity levels in the three color channels (red, green, blue), no saturated pixels could be observed.