Vectashield mounting medium with 4 6 diamino 2 phenylindole dapi

Cryo-sectioned 5 μm liver tissue slides were brought to room temperature and fixed with cold acetone for 8 min and then washed in PBS containing 0.05% Tween 20 (PBS-T). Nonspecific reactions were blocked with 5% normal goat serum (ab7481, Abcam, Cambridge, MA) and 5% TruStain fcX (BioLegend, San Diego, CA) in PBS-T for 1 h and then incubated with rabbit anti-mouse F4/80 (1:200, ab111101, Abcam) and rat anti-mouse Ly-6C (1:200, ab24973, Abcam) at 4°C overnight. After washing in PBS-T, the specimens were incubated with Alexa Fluor 488 goat anti-rat IgG (H+L) (1:400, #4416, Cell Signaling, Danvers, MA) and Alexa Fluor 555 goat anti-rabbit (1:400 #4413, Cell Signaling) for 1 hour at room temperature, washed again, treated with Vector TrueVIEW Autofluorescence Quenching Reagent and then counterstained with VECTASHIELD Mounting Medium with 4’,6-diamino-2-phenylindole (DAPI) (both: Vector Labs, Burlingame, CA). The Zeiss Axio Observer Microscope (Carl Zeiss Micro Imaging, Inc., Thornwood, NY) equipped with the Zen pro 2.3 software was used to visualize the immunofluorescence staining for F4/80 and Ly-6C, and nuclear localization was provided by DAPI. The negative controls were obtained by incubating sections with non-specific rat IgG or rabbit IgG as described above.

Slides containing frozen sections of skin biopsies from healthy controls (n = 3), At-Risk individuals (n = 3), and SLE patients (n = 3) were defrosted at room temperature and fixed in ice-cold methanol for 10 min at −20 ^oC and rehydrated in PBS for 5 min. Sections were then blocked in 10% fetal calf serum (FBS) blocking solution in 1 % PBS for 90 min at room temperature. This step was followed by incubation with rabbit monoclonal (ab81321) to BDCA-4 (Abcam) at 1:20 dilution of 1% FBS overnight at 4 °C. After washing twice in PBS for 5 min and an intermediate wash in PBS/Tween-20 (0.05%), sections were incubated with a PE-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) at a dilution of 1:100 with 1% FBS, for 1 h at room temperature. Slides were washed as above and were dried and mounted in Vectashield Mounting Medium with 4,6-diamino-2-phenylindole (DAPI) (Vector Laboratories). Finally, sections were viewed with a Leica DMIRB/E fluorescence microscope (Leica Microsystems Ltd).