Dapi prolong gold mounting medium

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.

DCs isolated from different strains of mice were grown on Lab-Tex chamber slides (Sigma-Alderich) as we described previously [24] (link). Briefly, cells were fixed by incubating slides in methanol for 10 min followed by acetone for 5 min at −20°C. Afterwards, slides were rinsed three times for 5 min each at ambient temperature in PBS containing 0.05% v/v Tween-20 (PBS-T). Slides were then blocked for 30 min at ambient temperature in PBS-T containing 1% w/v BSA. Rat anti-CD8α (clone 53–6.7, eBioscience, San Diego, CA), rat anti-CD4 (clone Gk1.5, eBioscience), rat anti-CD8β (clone YTS156.7.7, BioLegend), and hamster anti-CD11c (clone HL3, BD Biosciences) were used for IHC. Immunostaining was done using CD11c/CD4, CD11c/CD8α, or CD11c/CD8β antibody combinations and staining for 1 h at 25°C. After three rinses for 5 min each in PBS, slides were incubated for 1 h at 25°C with secondary antibodies labeled with anti-hamster FITC or anti-Rat TRITC (Invitrogen). Slides were washed three times with PBS, air-dried and mounted with Prolong Gold DAPI mounting medium (Invitrogen). Images were captured at 1024×1024 pixels (original magnification = 20X) in independent fluorescence channels using a Nikon C1 eclipse inverted confocal microscope.

RS cell monolayers grown on Lab-Tex chamber slides were infected with 1 or 10 PFU/cell of parental virus or recombinant HSV-CD80 for 12 and 24 hr. Slides were then blocked for 30 min at room temperature (RT) in PBS-T containing 1% w/v BSA (PBS-TB). Slides were washed, incubated for 1 hr at RT with anti-HSV-1 gC-FITC (Genway, #20-902-170310) antibody. In some experiments DCs isolated from WT, PD-L1^-/-, and PD-L2^-/- mice grown on Lab-Tex chamber slides were infected with 1 PFU/cell of HSV-CD80 or parental virus for 24 hr. Cells were fixed by incubating slides in methanol for 10 min followed by acetone for 5 min at -20×C. Afterwards, slides were rinsed 3× for 5 min each at RT in PBS containing 0.05% v/v Tween-20 (PBS-T). Slides were then blocked for 30 min at RT in PBS-T containing 1% w/v BSA (PBS-TB). Slides were washed, incubated for 1 hr at RT with anti-HSV-1 gC-FITC, anti-CD80, anti-PD-L1, anti-HVEM, and anti-nectin-1 antibodies (Genway, eBioscience, San Diego, CA; abcam, Cambridge, MA). Sections were washed 3× with PBS, air dried and mounted with Prolong Gold DAPI mounting medium (Invitrogen). Images were captured at 20× on independent fluorescence channels using a Nikon C1 eclipse inverted confocal microscope.