Filter set 38 he

Separate groups of 1-dpf embryos (n = 30) from hsp70-EGFP transgenic fish were exposed to DMSO (0.1%) or tBHQ (10 µM) for 4 hrs at 28°C. After exposure, embryos were washed and held for 4 additional hours in 0.3× Danieau's before being inspected for EGFP expression by fluorescence microscopy using an Axiovert 200 inverted microscope with Zeiss Filter Set 38 HE (489038; excitation BP 470/40, FT 495, emission BP 525/50). Another experiment was performed by exposing 4-dpf eleutheroembryos (n = 15) to DMSO (0.1%) or tBHQ (10 µM) for 4 hrs at 28°C. After exposure, eleutheroembryos were washed with 0.3× Danieau's and inspected by fluorescence microscopy at 4 hrs post-exposure (104 hpf) to assess EGFP expression.

The images of B. subtilis and S. pneumoniae VL451/MK359 were acquired on a Leica DMi8 microscope with a DFC9000 GT camera, a 100x/1.40 NA phase‐contrast objective (Leica) and a Lumencor SpectraX light engine with the following filter settings: GFP: SpectraX‐Quad cube (Chroma #89000) 470/24 excitation & 515/40 emission; RFP: SpectraX‐Quad cube (Chroma #89000) 575/35 excitation & 595/40 emission; far‐red (MitoTracker Red): Alexa 633 cube (Leica #11103136): 610/655 excitation & 620/720 emission.
S. aureus was imaged using a Zeiss AxioObserver with ZEN Blue software. Images were captured using an ORCA‐Flash4.0 V2 Digital CMOS camera (Hamamatsu Photonics), a 100x phase‐contrast objective. HPX 120 Illuminator (Zeiss) was used as a fluorescence light source. The following filter settings were used for GFP: filter set 38 HE (Zeiss) 470/40 excitation and 525/50 emission.
S. pneumoniae with DnaX‐GFP (strain VL369/RR23) and DnaX‐GFP/FtsZ‐RFP (strain VL469/MK396) were imaged as described before (van Raaphorst et al., 2017). In short, the cells were imaged on a Deltavision Elite microscope in HiLo mode using a 488 nm (GFP) and 568 (RFP) laser through a Quad cube (Chroma #89000), either every 20 s at 30°C for 1 hr or every 2 min at 37°C for 3 hr an 48 min.

The AbrNLPs were cloned into the pGWB441 vector to have a C-terminal yellow fluorescent protein (YFP) fusion using Gateway Cloning. Primary cultures of pGWB441-AbrNLP1 and pGWB441-AbrNLP2 grown overnight were used for inoculating secondary cultures, which were grown till they reached an OD₆₀₀ of 0.8–1. The pellets from the secondary cultures were resuspended in the resuspension solution (MES—10 mM, MgCl2—10 mM, and Acetosyringone—200 μM). The resuspended cultures were incubated at room temperature (25°C) for 3–4 h prior to infiltration in 5–6 weeks old N. benthamiana leaves. The subcellular localization of YFP-tagged proteins was examined 2–3 days post infiltration using a Carl Zeiss confocal microscope (LSM880). For visualization of the nucleus and cell wall, the leaves were immersed in 4′,6-diamidino-2-phenylindole (DAPI) solution (10 μg/ml) and Propidium Iodide solution (10 μg/ml) sequentially for 15 min in each solution before imaging. Excitation of the corresponding leaf areas took place at 514 nm for YFP (Filter Set 38 HE, Carl Zeiss AG), at 405 nm for DAPI (Filter Set 49 HE, Carl Zeiss AG), and at 561 nm for PI (Filter Set 43 HE, Carl Zeiss AG).

Real-time Ca²⁺ imaging was performed on MC soma using an epifluorescence microscope (Zeiss axioscope) with a 40× objective (Zeiss Plan-APOCHROMAT). Ca²⁺-induced fluorescence was detected with excitation (470/40 nm) and emission (dichroic mirror: 495; 525/50 nm) band pass filters (Zeiss filter set 38 HE). The illumination was produced by a white LED (Dual Port OptoLED, CAIRN, Faversham, UK) using the same illumination power for the different samples. Pictures were captured with a digital CCD camera (ORCA Flash 4.0, Hamamatsu Photonics, Maccy, France). Frames for time-lapse imaging were acquired (every 20 msec) using HCimage software (Hamamatsu Photonics, Massy, France) and analyzed using ImageJ software (NIH, Bethesda, MD). The Ca²⁺ probes Fura-2 (200 μmol/L) or Fura2-FF (400 μmol/L) were dissolved in the intracellular medium. After passing in whole-cell configuration, 10 (Fura2) or 20 min (Fura2-FF) was allowed to pass before image acquisition. The ΔF/F was calculated as follows: The basal fluorescence (F_bas) was obtained by averaging the neuronal fluorescence intensity of the 500 msec preceding MC intracellular stimulation. The frame fluorescence (F_fr) was the neuronal fluorescence intensity of the frame. Therefore, ΔF/F = (F_fr − F_bas)/F_fr. The data are expressed as the mean ± SEM.