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.
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