Briefly, cells were stained by direct immunofluorescence using monoclonal antibodies conjugated with fluorochromes FITC, Phycoerythrin, Pycoerythrin-cyanin 5 and ECD.
The cover slips were mounted onto glass slides using Mowiol and viewed by direct immunofluorescence using the Leica inverted confocal microscope (DM IRE2).
Canada), ILT3 (R&D Systems) and ILT4 (R&D Systems) expression was measured by direct immunofluorescence using conjugated antibodies, and the immunostained cells were analyzed on a FACSCaliber flow cytometer (Becton Dickinson, San Jose, CA) using CELLQuest software (Becton Dickinson).
The proportion of infected cells and the number of bacteria per cell was directly determined by direct immunofluorescence using fluorescein isotiocyanate-IgG anti-Brucella LPS as described elsewhere [39].
Focal adhesions were visualized by direct immunofluorescence using monoclonal anti-Vinculin-FITC antibody (F7053, Sigma).
EV recovered from ultracentrifugation were suspended in double filtered PBS and stained by direct immunofluorescence using monoclonal antibodies.
A phycoerythrin-conjugated (PE) antibody against CD14 (Imunotech, Marseille, France) recognising monocytes cells by direct immunofluorescence was used to make the distinction between cancer cells and monocytes.
Analysis of peripheral blood lymphocytes was performed by direct immunofluorescence flow cytometry using a Coulter EPICS ALTRA XL instrument.
Renal biopsy specimens were frozen and examined by direct immunofluorescence staining, performed using fluorescein-5-isothiocyanate-labeled rabbit anti-human IgG, IgA, IgM, C1q and C3 antisera (Dako, Denmark), goat anti-human C5 and P antisera (Nordic Immunological Laboratories, Tilburg, Netherlands), and rabbit anti-human fH antiserum labeled by Linkit™ Fluoro-Link (ISL, Paignton, UK).
Differentiation was assessed by direct immunofluorescence staining of cells using an allophycocyanin anti-human CD11b (Becton Dickinson, San Jose, CA, USA), PerCP anti-human CD14 (Becton Dickinson) and PE-IgG1 isotype control (eBiosciences, San Diego, CA, USA) as described previously.
Detection of apoptotic cardiac myocytes was achieved by direct immunofluorescence detection of digoxigenin-labeled genomic DNA using the ApopTag Plus in Situ apoptosis detection kit – Fluorescein (Intergen, Purchase, NY).
