Plan apochromat na1

HBMECs were seeded on Zeiss high–performance coverlips no. 1.5, infected with meningococci for 1 h, fixed in 4% paraformaldehyde for 10 min and labelled as described for STORM. Labelled preparations were mounted in DAKO Fluorescent Mounting Medium (S3023) on StarFrost 76 × 26 mm² microscope slides. SIM images were acquired on a Zeiss ElyraPS1 microscope with × 100 oil-immersion lens (Plan-Apochromat NA 1.46), and a resolution of 100 nm along the x–y-axis and 300 nm along the z-axis (Z-step of 0.116 nm). Super-resolution SIM images were obtained from 15 images (five different phases of three different angular orientations of the illumination pattern), collected on an EMCCD camera (1,004 × 1,002 pixels), processed with Zen (Zeiss) Software and analysed using Imaris 7.7 (Bitplane, Oxford Instruments).

Cells were sampled (200 µl) from the batch cultures at the mid-log phase and fixed in 4% PFA, stained with 0.1 µg µl⁻¹ of DAPI (Sigma-Aldrich, India) and mounted, as has been described previously [28 (link)]. Fixed cells of E.coli MG1655 recA-GFP and E. coli ΔrecA expressing RecA-Cherry were acquired using the 100× (Plan Apochromat N.A. 1.4, oil) objective of a Zeiss Axio Imager Z1 (Carl Zeiss, Germany) microscope in fluorescence and DIC channels. For live-imaging, cells were grown on 2% agar pads with 100 µg ml⁻¹ ampicillin and induced for 2 h by 0.2% Arabinose (Sisco Research Labs, Mumbai, India) to express HupA-GFP and imaged on a Zeiss LSM780 confocal microscope (Carl Zeiss, Germany) simultaneously in fluorescence (Diode laser 405 nm, beam splitter MBS 405, pinhole 126.5 corresponding to 1 airy unit) and DIC modes using a 63× lens (Plan Apochromat NA 1.40, oil). Multiple positions were scanned as 512 × 512 pixel images (0.264 µm per pixel) with an image acquired every 2 min for approximately 2 h and at 37°C.

HeLa cells were either mock transfected with water or transfected with VCIP135 siRNA duplex (30 nM) using TransIT-X2 (Mirus). After incubation for 46 h, the cells were fixed and used to observe the Golgi structures. For the rescue experiments, the mammalian expression construct (0.44 mg/l) of the HA-tagged rat VCIP135wt/mutant, which is insensitive to human VCIP135 siRNA, was transfected using TransIT-X2, 18 h after the treatment of cells with VCIP135 siRNA and the cells were further cultured for 28 h.
For the observation of Golgi structures, cells grown on coverslips were fixed with 3% PFA/PBS for 6 min, permeabilized with 0.1% T × 100 for 5 min, and stained with polyclonal antibodies to GM130 and monoclonal antibodies to HA-tag. The stained cells were observed using a confocal microscope (Zeiss LSM700) equipped with a 63× objective lens (Plan-Apochromat, NA 1.40).

Samples were illuminated by solid state lasers at 488 nm (Coherent Obis, 50 mW) with intensity 0.45 W/cm² and at 635 nm (Dragon laser, 300 mW) with intensity 40 W/cm². A custom dichroic (Chroma Technology, optimized for 488/640 illumination) within the CSU-X1 unit passed the excitation beams and reflected the sample fluorescence. Imaging was performed with a 20x water-immersion objective with coverslip correction (Zeiss Plan-Apochromat NA 1.0). Emission fluorescence passed through a quad-band emission filter (Chroma NC264505-ZET405/488/532/642m) and then passed through the spatial filter apparatus described above. A home-built dual-view imaging system projected emission of wavelengths <540 nm and >640 nm onto adjacent halves of an EMCCD camera (Andor iXon X3, 512 × 512 pixels). Exposure times ranged from 8 to 50 ms. A piezoelectric objective scanner (PIFOC PD72Z4CA0) set the z-position of the focus. A custom LabView (National Instruments) script controlled illumination, the objective scanner, and the camera for automated data acquisition.

Plants were imaged on a Zeiss 710 Confocal laser scanning microscope with a 20× Plan-Apochromat NA 1.0 water immersion lens. Note, none of our air lenses could achieve a high enough resolution to resolve the fluorescent signal. The mCitrine plasma membrane marker was excited with a 514 nm argon laser and emission spectra collected from 518 to 629 (for the experiment in Figs. 3 and 5A, B) or 519–650 nm (for the experiment in Figs. 5C and 7), through a 458/514/594 (for the experiments in Figs 3, and 5A, B) or 458/514 dichroic mirror at 1–2% detector gain (for the experiment in Figs. 5C, 6 and 7). If the plants could no longer be captured within one stack, the entire visible surface of the leaf was tiled over. Cellular landmarks were used to move the sample and create small areas of overlap to ensure that every section of the leaf surface of interest is captured. Within each tile, the z-range was adjusted to minimize time imaging the leaf. When tiled manually, the cellular landmarks that overlap were used to align stacks and assemble them in MorphoGraphX 2.0 [13 (link)].

Fixation of the incubated cells in the chamber slides was carried out for 10 min in an ice-cold mixture of 100% methanol and ethanol in a relation of 1:1. After air-drying the slides, blocking, and staining of the primary antibody with combinations of H3K4me3, H3K9ac, and PPARγ (for dilutions see Table 1) was performed as described for the double immunofluorescence staining of tissue slides. Since no unspecific staining could be determined within the HVT-cells respectively isolated EVT, a PPARγ antibody with a mouse host was used (Abnova, Taipeh, Taiwan—dilution 1:100).
The slides were washed with PBS between all individual steps. Secondary antibody staining and covering was performed as for the double immunofluorescence staining of tissue slides. Corresponding negative controls can be found in the supplement (Figure S7). Representative pictures for analysis were taken with a confocal laser scan microscope (LSM 510 Meta, Zeiss). LSM 510 Meta 18 confocal laser scanning microscope consists of an Axiovert 200M equipped with Differential Interference Contrast (DIC) and with a range of excitation laser lines: Ar diode laser: 405 nm (30 mW), Ar: 458, 477, 488, 514 nm (30 mW), HeNe: 543 nm (1 mW), HeNe: 633 nm (5 mW). Used lens for analysis: 63× Plan-Apochromat NA 1.4, d in mm 0.19 from Zeiss.