Mouse anti human cd61

Frozen sections of duodenal biopsy samples were stained with mouse anti-human CD36 (1∶200 final dilution, BD Bioscience, San Jose, CA) followed by incubation with alexafluor 488-conjugatedsecondary antibody (1∶400 final dilution, Life Technologies). Additional sections were fixed with paraformaldehyde and ethanol, and incubated with a mouse anti-human TSP-1 antibody (1∶100 final dilution, Novus Biologicals, Cambridge, UK) or mouse anti-human CD61 (1∶8000 final dilution, BD Bioscience, San Jose, CA) for 1 h at room temperature. Immunoreactive cells were visualized using Ultravision HRP-Polymer kit (Thermo Fisher Scientific, Waltham, MA) and DAB (Dako, Glostrup, Denmark), according to the manufacturer’s instructions, and lightly counterstained with hematoxylin. Control sections were prepared under identical immunohistochemical conditions replacing the primary antibody with isotype control antibodies (Dako).

Fluorescence microscopy was used to visualize the C. acnes-induced platelet aggregates. C. acnes was added to PRP as previously described. For C. acnes labeling DAPI and Oregon Green were used as indicated. PBS, PPP and PRP alone were used as negative controls, and collagen I as a positive control for platelet aggregation. The samples were fixed in 4% paraformaldehyde for 45 minutes and washed in D-PBS supplemented with 5% BSA and 50 mM glycine. Primary antibodies were added (mouse anti-human CD61 1:500, BD, Sparks, MD, USA) and incubated for 1 hour at room temperature, before washing in PBS + 5% BSA, and addition of secondary and/or labeled primary antibodies (Alexa Fluor 594 labeled anti-mouse IgG 1:500, Invitrogen, Grand Island, NY, USA; Alexa Fluor 647 labeled goat anti-human IgG 1:1000, Jackson ImmunoResearch, West Grove, PA, USA; FITC labeled rabbit anti-human fibrinogen 1:500, Dako, Santa Clara, CA, USA), continuing incubation for 1 hour. Finally, the samples were washed and re-suspended in PBS + 5% BSA and adsorbed on pre-coated poly-L-lysine coverslips for one hour before mounting to slides using DAPI ProLong Gold mounting medium (Invitrogen, Grand Island, NY, USA). The samples were allowed to settle overnight and analyzed in a Nikon Eclipse Ti microscope.