Plan apochromat 63x 1.4 oil

For immunofluorescence staining, cells were fixed for 5 min with ice cold methanol, additionally treated with 4% (w/v) paraformaldehyde for 10 min, blocked with 2% BSA (Carl Roth) in PBS, and incubated with the appropriate first (Sigma) and secondary (Invitrogen) antibodies. Nuclei were stained with DRAQ5. Cells were mounted in Mowiol 4-88 supplemented with 1% N-propyl gallate.
Confocal microscopy was carried out using a LSM 780 META microscope (Carl Zeiss) equipped with a Plan-Apochromat 63x/1.4 oil immersion objective. Total internal reflection fluorescence (TIRF) microscopy employed an Olympus IX71 TIRF Microscope (Olympus) and the MetaMorph software (Molecular Devices). Epifluorescence and TIRF images were acquired using the same exposure settings. S100A10 ablated cells were identified by epifluorescence. Quantitative evaluation of CCR10 mean gray values (TIRF field at 488 nm) of 162 cells transfected with non-targeting control siRNA and 144 cells transfected with S100A10 specific siRNA was performed using the ImageJ software (V 1.49a, National Institute of Health, USA). Mean values of empty areas were subtracted.

Bacterial labeling of EQKU cells was imaged on a Zeiss Elyra PS.1 microscope with Plan-Apochromat 63x/1.4 oil differential interference contrast (DIC) M27 objective. Excitation of Alk488 was achieved with 488 nm laser excitation, and the camera exposure time was set to 100.0 ms. The raw data contained five rotations with 0.110 μm z-stack interval. Images were processed in Carl Zeiss ZEN 2012 to construct SIM images. Processing and filtering settings were kept constant and image intensity was preserved using the raw image scale option. Two-dimensional (2D) SIM images and 2D maximum intensity projection images were generated.

A. nidulans strains were inoculated in liquid minimal media on sterile cover slides (18 × 18 mm, Th. Geyer, Höxter, Germany) that were placed in sterile petri dishes. In 400 µL medium, 2,000 spores were inoculated and grown for 20 h at 37 °C under illumination. Just before microscopy, the remaining medium was removed from the cover slide with a tissue. Twenty microliters of the fresh minimal medium were placed on an object slide, and the cover slide was placed on this fresh medium. The cover slide was fixed with nail polish. The inverted confocal microscope Zeiss AxioObserver, Z.1, which is equipped with Plan-Neofluar 63x/0.75 (air), Plan-Apochromat 63x/1.4 oil, Plan-Apochromat 100x/1.4 oil objectives (Zeiss, Oberkochen, Germany), and a QuantEM:512SC camera (Photometrics, Tucson, AZ, USA), was used in combination with the SlideBook 6.0 software package (Intelligent Imaging Innovations GmbH, Göttingen, Germany). Thereby, pictures were only taken with the Plan-Apochromat 100x/1.4 oil objectives (Zeiss, Oberkochen, Germany). The wild type strain AGB551 or the AGB1014, which expresses ^PgpdA:rfp:h2A, was included in the analyses for the normalization of GFP fluorescence.

Confocal microscopy experiments were performed by using Carl Zeiss LSM 510META instrument, with objective Plan-Apochromat 63x/1.4 Oil. The excitation was provided by 405 nm diode laser. The samples were located in humidified thermostated box at 37 °C.

Macrophages adhered on glass slides for 18 h at 37 °C were stimulated with M. bovis BCG-GFP (MOI:4) and with anti GM-CSF antibodies (Clone A7.39; 1 µg/mL) or IgG2b isotype control (1 µg/mL). After 24 hours, the slides were washed twice in PBS, fixed in PFA 4% for 15 min, and coverslips mounted using Fluoromount®. The cells were observed using a confocal microscope LSM510 Meta (Carl Zeiss SAS) with a Plan-Apochromat 63x/1.4 OIL objective. Images were processed using LSM510 Meta software and ImageJ.

A. nidulans colonies were visualized with an Olympus CS30 digital camera combined with an Olympus SZX-ILLB2-200 binocular. Pictures were processed with the cellSens software (Olympus). For fluorescence microscopy 500–2000 spores were inoculated in an 8-well borosilicate coverglass system (Thermo Scientific) containing 400 μl liquid minimal medium. Microscopy was performed with a Zeiss Axio Observer Z.1 system with Zeiss PlanAPOCHROMAT 63x/1.4oil. Pictures and movies were obtained with a QuantEM:512SC (Photometrics) or a Coolsnap HQ² (Photometrics) camera and the SlideBook 5.0 imaging software (Intelligent Imaging Innovations Inc.). Membranes were stained with 1 mM FM4-64 (Invitrogen) and mitochondria with 50 nM MitoTracker Red CMXRos (Invitrogen). Nuclei were visualized with ectopically integrated h2A::rfp. Exposure times for GFP: 1000 ms, RFP: 100 ms, DIC: 100 ms and in BiFC experiments exposure times were YFP: 1000 ms, RFP: 100 ms, DIC: 100 ms.

Cells were plated on poly‐D‐lysine (Sigma) coated glass. Then cells were fixed with paraformaldehyde 5%, permeabilized with PBS 0.3% Triton, blocked with PBS 5% Bovine Serum Albumin (SERVA) and immunostained with the indicated primary antibody. Signals were revealed using fluorescent‐ or rhodamine conjugated secondary antibodies (1:200; Invitrogen). Nuclei were stained with the cell permeable DNA fluorescent dye DRAQ5™ (Abcam ab108410). For cilia counting, we considered as cut‐off for cilia the length ≥ 1 μm. Immunostaining was visualized using a Zeiss LSM 510 META laser scanning confocal microscope. Where indicated, high‐resolution images were acquired with a Zeiss LSM 880 confocal microscope equipped with a Plan‐Apochromat 63x/1.4 oil immersion objective. Where indicated, high‐resolution images were acquired with a Zeiss LSM 880 confocal microscope equipped with Airyscan super‐resolution imaging module, using a Å ~ 63/1.40 NA Plan‐Apochromat Oil DIC M27 objective lens (Zeiss MicroImaging, Jena, Germany).