Lms 710 confocal microscopy

The right lungs were fixed in 4% PFA in PBS for 72 h and embedded in paraffin. One section was stained with hematoxylin and eosin (H&E) and examined in fifteen fields of views at a 10-fold magnification using IX73 microscopy (OLYMPUS). The other section was used for Immunofluorescence staining using anti-CD68 antibody [FA-11] (Alexa Fluor^®647) and examined by LMS 710 confocal microscopy (Zeiss).

PA was heat-killed at 60 °C for 2 h. Bacteria were fluorescently labeled using FITC (HOOK^TM) Dye Labeling Kit in the dark for 1 h on the shaking table. The labeled bacteria were washed twice in PBS to remove unbound label and re-suspended in PBS. Fluorescent-labeled bacteria were added to cells and incubated for 2 h.
Cells were seeded in confocal dish for at least 12 h and FITC-label PA (MOI 100:1 or MOI 300:1) were added to incubate for 2 h. Cells were washed with HBSS three times and fluorescence of extracellular particles was quenched by adding trypan blue (2% (volume/volume)) for 1 min. the sub-cellular localization of FITC-label bacteria was monitored under a LMS 710 confocal microscopy (Zeiss). The phagocytosis was investigated by IX73 microscopy (OLYMPUS) and quantified by C6 flow cytometry (BD Biosciences).