Ix81 confocal microscope

ALI cultures treated with EGF for 24 h were fixed in a 50:50 mixture of methanol-acetone, permeabilized with 0.3% triton X-100, and blocked with 5% skimmed milk. Specimens were incubated overnight at 4 °C with TMEM16A antibody (1:200, Abcam) and MUC5AC antibody (1:200, Abcam), and then further incubated with secondary antibody rhodamine-conjugated goat anti-mouse IgG (1:500, Invitrogen) and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:500, Invitrogen). All specimens were then counterstained with 4, 6-diamidino-2-phenylinodole nuclear stain and examined by microscopy using an Olympus IX 81 confocal microscope (Tokyo, Japan). The number of TMEM16A-positive cells, MUC5AC-positive cells, and cells coexpressing TMEM16A and MUC5AC in 10 random fields of vision per culture was counted and expressed at a percentage of total cells.

RED FLOURESCENT PROTEIN (RFP) expression was visualised using a Leica Fluo III fluorescence microscope (Leica, Wetzlar, Germany) and an Olympus IX81 confocal microscope (Olympus, Tokyo, Japan) equipped with an RFP filter set (excitation wavelength 580 nm and emission wavelength 630 nm). For visualisation of RFP fluorescence in callus material 5-days post co-cultivation with Agrobacterium, callus remained in sealed culture plates, however, RFP expression was visualised in internode and root samples via the mounting of these plant materials on glass slides. Wild-type material that had not been co-cultivated with Agrobacterium harbouring the pANIC12A vector, was used as the negative control for RFP investigation. All images were collected under bright field illumination, and via the use of the same RFP filter set and microscopy settings.

To study hCD81 variant expression patterns, immunofluorescence staining followed by confocal microscopy was performed. To that end, hCD81 variant-expressing Lunet N#3 cells were seeded on poly-l-Lysin-coated cover slips in 24-well plate at a density of 5 × 10⁴ cell/well and incubated at 37 °C. After 24 h of incubation, cells were fixed for 20 min with 3% paraformaldehyde (PFA) at room temperature followed by washing with PBS. The cells were permeabilized using 0.1% Triton X-100 in PBS for 5 min. Afterwards, the cover slips were blocked with PBS/0.5% BSA for 10 min and incubated at 4 °C overnight in the presence of primary antibody (Mouse anti-human CD81, BD clone JS-81, 10 μg/ml, diluted 1:50 in PBS/BSA). Unbound primary antibody was washed off with PBS and the cells incubated with secondary antibody (goat anti-mouse-IgG-Alexa 488 diluted 1:1000 in PBS/BSA) for 1 h at room temperature in the dark. Following a washing step, nuclei were stained with DAPI (diluted 1:10,000 in H₂O) for 1 min in the dark. Residual dye was removed by washing the coverslips with H₂O. The cover slips were then mounted on glass slides using 7 μl of ProLong ^® Gold Antifade (Thermo Fischer). Immunofluorescence analyses were carried out using inverted Olympus IX-81 confocal microscope and the FV1000 Viewer (Olympus).

For live cell imaging, infected cells were resuspended in filtrated HL5-C and seeded in μ-Slide 8 well chambers (Ibidi) shortly before imaging. Microscopic analysis was performed at 21°C using a Zeiss LSM5 Live confocal microscope with a 100x Europlan apochromat oil immersion objective (N.A. 1.4) and a Diode-Laser 488, as well as a DPSS-Laser 561 [single track mode, 1 Airy unit, dual-band filter (500–545 band pass, 575 long pass)]. Image brightness and contrast were adjusted with ImageJ (Schneider et al., 2012 (link)) to whole images. A minimum of 100 bacteria were quantified at each timepoint except for F.n.n. ΔiglC at 2 hpi (50 bacteria). Imaging of PFA-fixed samples was performed with an Olympus IX81 confocal microscope equipped with an Olympus 100x UPlanSApo oil immersion objective (N.A. 1.4).

Flight muscles were dissected in cold PBS and fixed in freshly made 4% formaldehyde for 15 minutes. After 10 minutes washing in PBS containing 0.4% (v/v) Triton X-100 (PBTX) 3 times, the muscles were incubated with ATP5α Abs diluted in 0.4% PBTX containing 5% goat serum overnight at 4°C. The muscles were then incubated at room temperature with conjugated secondary Abs for 2 hours, followed by staining with DAPI for 10 minutes. After washing, the muscles were mounted on glass slides with VECTASHIELD Antifade Mounting Medium (Vector Laboratories) and kept at 4°C until imaging. Specimens were imaged using an Olympus IX81 confocal microscope. Images were processed using FluoView 10-ASW software (Olympus) and analyzed using ImageJ software.

ROS generation was detected as previously described22 (link). An Olympus IX81 confocal microscope and an Olympus oil immersion objective lens (40x/1.3 Plan-Apochromat) (Olympus, Milan, Italy, EU) were used. The images were acquired (1 frame every 30 seconds, 140 frames total) and processed using Xcellence software (Olympus).