Mouse anti cd45

Formalin fixed paraffin-embedded (FFPE) skin specimens from EV-lesions were obtained from the Department of Dermatology, Medical University of Warsaw, Warsaw, Poland. The presence of HPV8 was confirmed by quantitative real-time PCR (qRT-PCR) as described in Weissenborn et al. (2010) (link). Sections were stained with mouse monoclonal anti-CD15 antibody (clone Carb-3, Dako, Glostrup, Denmark), rabbit monoclonal anti-S100A8 antibody (clone EPR3554; Novus Biologicals, Cambridge, United Kingdom), rabbit polyclonal anti-S100A9 antibody (H-90, sc-20173, Santa Cruz Biotechnology, Heidelberg, Germany) or mouse anti-CD45 (Abcam, Cambridge, United Kingdom). Staining was performed using the Dako instrument Autostainer Plus (Dako) or Immpress AP Reagent kit (Vector, Burlingame, CA, United States). For immunofluorescence, cells were grown on cover slips, fixed with 2% buffered paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained with rabbit anti-HPV8 E2 (from Mart Ustav, University of Tartu, Tartu, Estonia) and mouse monoclonal anti-pan-cytokeratin (clone C11; Sigma-Aldrich, Steinheim, Germany) and secondary goat anti-rabbit Alexa Fluor 546 goat and goat anti-mouse Alexa Fluor 488 (Life Technologies, Eugene, OR, United States), respectively.

Subclassification of the CD90⁺ population was investigated by the double immunostaining of CD90 with CD24 (CSC marker for PDAC), and cellular markers including α-smooth muscle actin (αSMA, a marker of activated pancreatic stellate cells), CD31 (endothelial vascular cell marker) and CD45 (leukocyte common antigen), respectively. The TMAs were dewaxed, rehydrated, and treated with citrate buffer for antigen retrieval and then 2% BSA to block non-specific binding as described above. To achieve double immunofluorescence (IF) staining of CD90 and the known markers, rabbit anti-CD90 (Abcam, Cambridge, MA) was mixed with mouse anti-CD24 (Abcam, Cambridge, MA), mouse anti-αSMA (Sigma), mouse anti-CD31 (Novocastra, Newcastle Upon Tyne, UK), and mouse anti-CD45 (Abcam, Cambridge, MA) antibodies, respectively, at the optimal dilutions according to the manufacturer's instructions. The antibody mixture was then incubated with the TMAs overnight at 4°C. Then DyLight 549 anti-mouse IgG (red) and DyLight 488 anti-rabbit IgG (green) (Vector laboratories, Burlingame, CA) were diluted (1∶200) and incubated with the TMAs for 1 hr at room temperature. Nuclei visualization was explored by DAPI counterstaining (blue). The TMAs were finally dehydrated in alcohol and coverslipped.