H 1200

The IF was performed on 10% neutral-buffered formalin-fixed cells in 8-chamber slides. The fixed cells were then permeabilized with 0.1% Triton-X 100 in PBS for 15 min at RT, washed twice before blocking with protein blocker (Dako) for 20 min, Fc receptor blocker (Innovec) for 15 min followed by 30 min incubation with normal horse serum (Vector). Primary antibody was mouse anti-IE (Millipore, cat. no. MAB810R, 1:500) and rabbit anti-ET-1 (Abcam, cat. no. ab170544, 1:100; targeting the preproET-1, 183–211 aa). For dual-immunofluorescent staining, both primary antibodies were added together and incubated at 4 °C for 16–18-h. Secondary antibody goat anti-mouse Alexa Fluor 488 (cat. no. A11001) and goat anti-rabbit Alexa Fluor 594 (cat. no. A11012) were added together and incubated for 1-h at RT. All the secondary antibodies were from the Molecular Probes, Invitrogen and used at 1:500 dilutions. Nuclei were counterstained with 4′-6-Diamidino-2-phenylindole provided with the mounting medium (Vector, H-1200) and slides were viewed under Leica confocal microscope and analyzed with Leica Application Suite Advanced Fluorescence software or Zeiss LSM 700 Confocal with Zen software.

The cells expressing CD19-GFP fusion variants were fixed for 10 min on ice with 4% paraformaldehyde, washed, and then stained with 5 μg/ml wheat germ agglutinin-Alexa Fluor 680 (Molecular Probes; W32465) according to the manufacturer's instructions. The cells were permeabilized with 0.2% saponin, 0.5% bovine serum albumin (BSA), 0.03 M sucrose in phosphate-buffered saline (PBS) for 10 min at room temperature. Primary calnexin antibody was incubated overnight at 4°C. Secondary Alexa Fluor 594–anti-rabbit antibody was incubated for 1 h at room temperature. The cells were the washed and mounted on precharged glass microscope slides with DAPI (4′,6-diamidino-2-phenylindole)-containing medium (Vectashield; H1200) and visualized under a Leica STED 3× superresolution confocal system (HC PL APO CS2 63×/1.40-numerical-aperture oil 63× objective). Images were acquired using 4,184-by-4,184 resolution with limited signal saturation. Colocalization was quantified by Pearson correlation coefficient. Six images for each CD19 construct containing 100 cells on average were analyzed with BioImageXD and Fiji Coloc2 plug-in software. The statistical Costes P value was 1 for this analysis (38 (link)).

To confirm dsRed/EGFP expression, cells were plated onto poly-lysine coated chamber slides (Thermoscientific Nunc Lab-Tek), allowed to attach for 1–2 hours, fixed with 4% paraformaldehyde in PBS, pH 7.0, washed and immunostained with rabbit Anti-dsRed (632496, Clontech; 1:1000) and/or mouse Anti-EGFP (DHSB 12A6) antibodies and the respective Alexa dye-coupled secondary antibodies (Jackson ImmunoResearch) reconstituted in 5% Normal Goat Serum, 0.1% Triton in PBS. Cells were then mounted on DAPI containing mounting media (Vectashield H-1200) prior to imaging on a Leica SP8 confocal microscope.