Antigen retrieval solution

The paraffin-embedded sections were diagnosed by dermatopathologists (M.W. or H.K.). Additional immunohistochemistry for PRAME was assessed by H.K. and V.G., a resident physician with special interest in dermatopathology. Immunohistochemistry for PRAME was performed on 5 µm tissue sections of the primary tumors and, if available, associated metastases, which were deparaffinized in xylol and rehydrated in graded alcohol before. The slides were overlaid with antigen retrieval solution (Dako, Hamburg, Germany) and incubated in saturated steam for 30 min. For staining, slides were incubated with an anti-PRAME antibody (MAb EPR20330; Abcam, #219650, 1:1000) using the Dako Autostainer plus (Dako) and the Dako REAL Detection System Alkaline Phosphatase/ RED (#K5005Dako, Dako). For PRAME and melanocytic markers (S100, MART-1, SOX-10 and HMB-45), we assessed nuclear and cytoplasmatic patterns of staining. Focal positivity was marked when only some areas of the tumor stained positive.

Five-micrometer serial sections from paraffin blocks of mouse brain samples were collected with a microtome. To visualize the Aβ plaques in mice an immunostaining with Aβ-6E10 and with the Thioflavine-S was performed. After rehydration, slices were incubated in the antigen retrieval solution (DAKO, Glostrup, Denmark) at 95 °C for 25 minutes followed by the blocking solution (7.5% NGS; 0.4% Triton; 1% BSA; PBS) for 2 h and in Aβ-6E10 antibody solution (5% NGS; 0.4% Triton; PBS) overnight at 4 °C. Slices were incubated 2 h 30 minutes at RT in the secondary antibody solution and 5 minutes in 30 nM DAPI. For the Thioflavine-S, slices were rehydrated, incubated in Thioflavine-S 0,1% for 3 minutes, rinsed with EtOH 70% and cover-slipped with Fluoromount mounting media. As a control for specificity of antibodies, some sections were treated as described except that the primary antibody was omitted from the incubation medium. Slices were observed using a Zeiss AxioImager M2 microscope and images were processed with a computerized image analysis system (ZEN 2012 SP2 Software, Zeiss).

All tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin, and sections (5-µm thickness) were stained with hematoxylin and eosin or processed for antibody (Ab) staining. For IHC staining, sections were deparaffinized and treated with an antigen-retrieval solution (Dako, Carpinteria, CA) at 98°C for 25 min, a peroxidase-blocking solution (Dako) for 15 min, and then with a streptavidin-biotin blocking solution (Vector Laboratories, Burlingame, CA) for 1 h. Sections were incubated for 2 h at room temperature with one of the following antibodies: rabbit anti-O. tsutsugamushi Karp strain polyclonal antibody (pAb, 1∶500); rat anti-mouse neutrophil monoclonal antibody (mAb, 1∶25, clone 7/4, Caltag Laboratories, Buckingham, UK); rabbit anti- myeloperoxidase (MPO) pAb (1∶25, Abcam); or rabbit anti-mouse CD3 mAb (1∶25, Abcam, Cambridge, MA). Biotinylated goat anti-rat or anti-rabbit secondary antibodies (1∶200, Vector) were incubated on the sections for 30 min. Sections were stained with alkaline phosphatase-conjugated streptavidin (1∶200, Vector), Vector Red alkaline phosphatase substrate, and counterstained with hematoxylin (Sigma, St. Louis, MO). Reagent negative controls consisted of samples in which primary Ab was replaced with normal rat or rabbit IgG. Sections were dehydrated, mounted in Permount (Vector), and imaged under an Olympus BX53 microscope.

Ace-1-Vector and Ace-1-Dkk-1 were grown to 70% confluency, trypsinized, and centrifuged at 1000 RPM for 5 min. The cell pellet was then fixed in 10% neutral-buffered formalin for 24 h and embedded in paraffin. The specimens were sectioned (5 μm). Then they were deparaffinized, rehydrated, and incubated in antigen retrieval solution (Dako, Carpinteria, CA, USA) at 90 °C for 30 min before cooling down at room temperature for 15 min. The endogenous peroxidase was blocked using 3% hydrogen peroxide and serum-free protein block (Dako). Samples were incubated at room temperature for 1 h with monoclonal mouse anti-β-catenin (BD Pharmingen, San Jose, CA, USA, Cat. No. 610153, 1:250) in phosphate-buffered saline (PBS). Anti-mouse monoclonal (Vector Laboratories, Burlingame, CA, USA, Cat. No. BA-9200, 1:200) in PBS was applied as the secondary antibody. Slides were stained with a Vector ABC Elite complex (Dako) for 30 min, diaminobenzidine tetrahydrochloride (Dako) for 5 min, and Mayer’s hematoxylin for 1 min. Image Pro Plus 9.0 (Media Cybernetics, Rockville, MD, USA) was used to perform batch analysis and quantify total cell staining (reported as optical density, OD). The values from 5 fields of approximately 700 cells were averaged.

Formalin fixed and paraffin embedded pancreas tumors and their corresponding metastatic liver lesion were sectioned and then analyzed with the Nanostring GeoMx DSP at the MUSC Translational Science Laboratory. Slides were processed as per the GeoMx DSP slide prep protocol. In brief, deparaffinized slides were subjected to antigen retrieval with DAKO antigen retrieval solution and blocked 1 h in Buffer W. Slides were hybridized with oligonucleotide labeled antibody probes from the Immune Cell Profiling Panel Mouse Protein Core and fluorescently stained with three morphology markers targeting macrophages (F4/80), total immune cells (CD45), and tumor bed (pan cytokeratin CK) as well as a nuclear stain (SYTO 13). Regions of interest (ROI) were circled based on areas positive for all three of these morphology markers. Oligos encircled in each ROI were cleaved and released after being targeted by UV light. The oligos were collected in a 96 well plate and hybridized to barcodes before being counted on the nCounter platform. Quality control, normalization and heat map generation were all performed using the GeoMx software as recommended from Nanostring.