Zen 2012 black software

Cells grown on sterilized cover slips were fixed with 4% paraformaldehyde for 15 min at room temperature; permeabilized with 0.1% Triton X-100 for 5 min at room temperature; and then incubated for 1 h in blocking buffer (2% BSA, 5% glycerol, 0.2% Tween20, 0.1% NaN₃). The cover slips were subsequently incubated with primary antibodies for 1 h at room temperature, followed by 30 min with the secondary antibody, both diluted in blocking buffer. After mounting with Vectashield medium containing DAPI (Vector Laboratories, Bionordika), images were acquired with an LSM700 confocal microscope (Zeiss) mounted on a Zeiss Axio observer.Z1 equipped with Plan-Apochromat ×63/1.4 oil immersion lenses and subsequently processed utilizing the Zen 2012 Black software (Zeiss).

Images were acquired with an LSM700 confocal microscope (Zeiss), mounted on an Axio Observer.Z1 (Zeiss) equipped with a Plan-Apochromat 63×/1.4 oil immersion lens. For images that were not to be analyzed, Z-stacks were acquired and processed with the Zen 2012 Black software (Zeiss) to produce maximum intensity projection images. For analysis of the intensity of signals in speckles, single-plane images were acquired on the focal plane that showed the highest intensity. Image analysis was performed in CellProfiler (v3.1.9) using a custom pipeline. Data analysis was performed using R (v4.1.1). Intensity profiles over speckles were generated using Fiji (v2.3.0).

Salmonella transformed with the eGFP-pBAD plasmid (a gift from Michael Davidson, Addgene plasmid #54762; http://n2t.net/addgene:54762; RRID:Addgene_54762) was used to confirm intracellular localization. Images of infected cells were acquired every hour using a Zeiss LSM 780 laser scanning confocal microscope equipped with a Plan-Apochromat 40×/1.3 Oil DIC or Plan-Apochromat 63×/1.4 Oil DIC objective (Zeiss, Jena, Germany). Single mages and time-lapse series were acquired using the ZEN 2012 Black software (Zeiss). Differential interference contrast was used for visualization of living cells on a TPMT detector. GFP fluorescence in Salmonella was quantitated by excitation at 488 nm with the argon laser and an emission at 490–569 nm in the Channel mode.

Tile scan images of fluorescently labeled sections were collected on a Zeiss LSM 780 or 880 inverted confocal microscope (Carl Zeiss Microscopy, Thornwood, NY), and z-stacks were converted to maximum intensity projections using Zen 2012 Black Software (Carl Zeiss). Images were modified only by using Photoshop (Adobe Systems, San Jose, CA) or ImageJ software (US National Institutes of Health) to adjust brightness and contrast across the entire image. Tile scan images of tissue cleared by PACT were collected on an LSM 880 confocal microscope using an EC Plan-Neofluar 10x/0.3 M27 objective (Carl Zeiss). Due to the thickness of the samples, the Auto Z Brightness Correction was used for both the 488 nm laser line from an Argon laser (2–20% power range) and 561 nm laser line from a DPSS laser (2–12% power range). The pinhole was set to yield an optical z-thickness of 14 μm, and a z-stack was collected at 10 μm interval between images. z-stacks were viewed with Imaris Software (Bitplane, Concord MA) for three-dimensional rendering of the entire region and surface rendering of individual cells.

S. Typhimurium 14028s and E. coli B/r were transformed by electroporation with plasmid pFCcGi (Addgene plasmid number 59324), encoding the red fluorescent protein mCherry expressed constitutively (Figueira et al., 2013 (link)). Axenic D. discoideum AX2 cnxA-GFP (∼2 × 10⁷ cells) was co-incubated with each bacteria at 22°C for 24 h in 10 mL of Soerensen buffer, using a MOI of 10 bacteria/amoeba. Images of infected cells were acquired every hour using a Zeiss LSM 710 laser scanning confocal microscope equipped with a 63x 1.4 NA optic setup. Prior to observation, cells were mounted on a thin layer of 1% agarose in PBS buffer deposited on a glass slide. To visualize GFP-associated fluorescence (amoebae), the sample was excited at 488 nm with an argon laser and emission was detected using a filter in the 493–549 nm range. To visualize mCherry-associated fluorescence (bacteria), the sample was excited at 543 nm with a HeNe laser and emission was detected using a filter in the 548–679 nm range. Images were acquired using the ZEN 2012 Black software (Zeiss), and analyzed using Fiji and ImageJ softwares (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)).