Ultravision lp detection system

Ventana BenchMark Ultra automated staining system (Roche Tissue Diagnostics, Grenzach-Vyhlen, Germany) was used for immunohistochemistry. From the formalin-fixed and paraffin embedded (FFPE) tumor specimen 3 μm sections were prepared. The following primary antibodies were used: anti-vimentin (Dako #M0725), anti-TTF1 (Dako, clone 8G7G3/1), anti-PAX8 (Cell Marque, MRQ-50), Cytokeratin 18 (Dako, Clone DC 10) and anti-thyroglobulin (Dako, A0251) and anti-PD-L1 (22C3, Dako). Antibody binding was detected with the UltraVision LP Detection System (Lab Vision Corporation). Color development with OptiView staining kit (Roche Tissue Diagnostics, Grenzach-Vyhlen, Germany) was followed by hematoxylin counterstaining. PD-L1 expression was quantified by a pathologist using the combined positive score (CPS).

To obtain information about C4d presence within the tumor tissue and to compare these tissue levels with circulating C4d levels, 32 FFPE MPM tissue specimens were collected and analyzed by C4d immunohistochemical staining. Additionally, we performed immunohistochemical staining for C1q in 14 FFPE tissue specimens deriving from patients with high (n = 7) and low (n = 7) circulating C4d levels. After the sections were deparaffinized and rehydrated, endogenous peroxidase activity was blocked with 0.3% H2O2 in phosphate buffered saline. Heat-induced epitope retrieval was performed by using TRIS/EDTA buffer (pH = 9). Primary antibodies (anti-human C4d; Biomedica, Vienna, Austria and anti-human C1q; ab71089, Abcam, Cambridge, UK) were diluted 1:500 and 1:1000, respectively, and incubated for 60 min at RT. Antibody binding was detected with the UltraVision LP Detection System (Lab Vision Corporation, Fremont, CA, USA). Color development was achieved by DAB followed by hematoxylin counterstaining. Stainings were evaluated by a pathologist.

Ki67 immunohistochemistry was performed as described earlier [33 (link)]. Three-micrometer sections from formalin-fixed and paraffin-embedded (FFPE) tumor nodules were deparaffinized and rehydrated with decreasing alcohol concentrations. Following heat-induced antigen retrieval in citrate buffer (pH 6.0), slides were incubated with the Ki67 antibody (mouse mAb, Dako Cytomation, clone MIB-1, dilution 1:100) for 30 min. UltraVision LP detection system (Lab Vision Corporation, Fremont, CA) was used to detect antibody binding according to the manufacturer’s recommendations. 3-3-Diaminobenzidine (Dako) was applied for color development. Each analyzed slide was counterstained with Mayer’s hematoxylin (Sigma) and images were taken using a bright-field microscope (Axio Imager, Carl Zeiss). Labeling index was determined by using the automated image analysis application ImmunoRatio [34 (link)]. At least 2000 tumor cells were counted to obtain the mean percentage of Ki67-positive MPM tumor cells per sample.

Expression of HBD-1 in prostate cancer tissue samples was studied using the UltraVision LP Detection System (Lab Vision Corp., Fremont, CA, USA), as described previously[27 (link)]. Briefly, slides with prostate cancer tissue sections were dewaxed in xylene for more than 20 min and then sequentially rehydrated in 100%, 95%, 90%, and 80% ethanol solutions. After a 5-min rinse in water, the slides were pretreated with 0.01M sodium citrate buffer and autoclaved for 1 min at 121°C for antigen retrieval. After the slides were rinsed, endogenous peroxidase activity was blocked by treatment with 3% H₂O₂ for 30 min. A primary mouse polyclonal antibody against HBD-1 (1:50, Abcam, Cambridge, MA, USA) was applied to the sections on the slides, following which the sections were incubated for 2 h in a moist chamber at room temperature. Sections for negative control were incubated with the same reagents and underwent the same epitope retrieval protocol without the primary antibody (S1 Fig). After the slides were rinsed with PBS, they were incubated with secondary antibody for 10 min at room temperature and then rinsed in PBS. The slides were incubated with a horseradish peroxidase conjugated antibody for 10 min, rinsed in PBS, and incubated with diaminobenzidine for 10 min. After the slides were counterstained using Mayer’s hematoxylin, they were dehydrated and coverslipped.