Immunofluorescence.
SECONDARY
Fresh tissues were obtained from the patients via 4–6 mm punch biopsy. Half of the tissues were fixed in 1% paraformaldehyde for 4 hours at room temperature and incubated in 20% sucrose overnight at 4°C. The tissues were sectioned (4 μm) and permeabilized with 0.5% Triton X-100 for 30 minutes. After washing with PBS, the slides were incubated in blocking solution (X0909, DAKO) for 1 hour. Tissues were stained overnight at 4°C using the following primary antibodies: mouse anti-human CD20 (L26, Abcam); 6X His-recombinant human DSG1 and 6X His-recombinant human DSG3 (both from Cusabio); goat anti-human CXCL13 (R&D Systems); rabbit anti-human CCL5 (P230E, Thermo Fisher Scientific); rabbit anti-human CD138 (EP201), rabbit anti-human FDC (CNA.42), and mouse anti-human CD8 (C8/144B) (all from Cell Marque); rat anti-human CD4 (YNB 46.1.8, Santa Cruz); and mouse anti-human HLA-DR (L243, BioLegend). Tissues were washed with PBS and incubated for 1 hour at room temperature with secondary antibody: Alexa Flour 488–conjugated goat anti-mouse and anti-rat antibodies; Alexa Flour 594–conjugated goat anti-rabbit, anti-mouse, and anti-rat antibodies; Alexa Flour 647–conjugated goat anti-mouse and anti-rat antibodies (all from Invitrogen); donkey anti-goat antibody (Jackson ImmunoResearch); and mouse anti-6X His-tag antibody (R&D Systems). Nuclei were stained with DAPI (Thermo Fisher Scientific). Fluorescence images were captured on an LSM 780 confocal microscope (Carl Zeiss).