Plan apochromat 20x objective

Lung tissues were fixed in 4% paraformaldehyde (PFA) (Sigma Aldrich, St Louis, MO) and then sequentially incubated at 4°C with 5%, 10% and 20% sucrose over a one-week period. Lungs were embedded in tissue-tek (OCT^®) and stored at -80°C. 10 μm lung sections were cut on a cryostat (Leica, Solms, Germany) and heated at 80° C for 30 min in 10 mM citrate pH = 6. Lung cells were permeabilized with 0.5% Triton X-100, blocked in 1% BSA, 10% FCS, 0.1% Triton X-100 in PBS for 1h, washed three times in TBS and incubated overnight with primary antibodies (see Supplementary Table 1 for details) in PBS containing 2% BSA, 10% FCS and 0.5% Triton X-100 at 4°C. Lung sections were washed with TBS and incubated with relevant secondary antibodies conjugated with Alexa Fluor dyes for 1h at RT. After washing, cells were counterstained with DAPI for 10 min at RT, washed with PBS and mounted onto microscope slides (mowiol). Slides were visualized using a Zeiss Axio Observer Z7 microscope coupled with LSM 980 AiryScan2 module device (Carl Zeiss Co. Ltd., Jena, Germany). Axio Observer Z7 microscope (Carl Zeiss Co. Ltd., Jena, Germany) inverted microscope was equipped with a Plan-Apochromat 20X objective (NA = 0.8). Images were acquired using ZEN v2.3 pro Zeiss software (Carl Zeiss Co. Ltd., Jena, Germany).

Confocal microscopy was performed using a Zeiss LSM 510 (Zeiss, Jena, Germany) confocal laser scanning microscope with a LD LCI Plan-Apochromat 25x water immersion objective (NA-0.8), or LSM 710 (Zeiss, Jena, Germany) using a Plan-Apochromat 20x objective (NA-0.8). Roots were imaged in water, or with water supplemented with propidium iodide (PI, 10 μg/mL). The green fluorescent proteins NeonGreen (NG) and PI were excited by an argon laser (488 nm) and by DPSS laser (561 nm), respectively. For PI detection in LSM 710, solid state laser (543 nm) was used. In LSM 510, fluorescence emission signals for NG and for PI were collected by PMT detectors, with a band-pass filter (500–530 nm) and a long-pass filter (575 nm), respectively. In LSM 710, fluorescence emission signals for NG and for PI were collected by BIG detectors (GaAsP) with a band-pass filter (500–550 nm) and a band-pass filter (570–620 nm), respectively.

CFSE Fluorescent Cell labelling (Abcam) was performed according to the manufacturer’s description before plating the cells in 15μ-slide 8 well plates (IBIDI) using DMEM without phenol red (Gibco). After overnight incubation, cells were infected with Lgy parasites and Dihydrorhodamine 123 (DHR, Invitrogen) was added simultaneously. Live cell imaging was performed on LSM800 confocal microscope (Zeiss) using Plan Apochromat 20x objective. Images were acquired with the pinhole totally open to scan in non-confocal mode in order to minimize the laser intensity on the cells. Six images per condition were acquired every 20 min during 14 hrs at 35°C and 5% CO₂ using full enclosure PECON incubator using Zen Blue (Zeiss) software. ROS level accumulation in BMDMs were quantified using Image J (NIH) by encoding a macro in RGB batch mode. Threshold was set for CFSE staining, which was used to define the ROI for measuring the cellular DHR signal as a proxy of ROS production. Results are expressed as Average intensity showing the mean of all the positions per condition at each time-point. Additionally, cell count was determined with CFSE staining and it is expressed as the mean of all the positions per condition at each time-point. All reagents used for ROS production measurement are detailed in S2 Table.