Imager m2 microscope

Cells were plated on coverslips and transfected with the indicated plasmids. After 24–48 h, cells were washed with PBS, fixed with 3.7% formaldehyde in PBS for 20 min, then permeabilized with 0.1% Triton-X100 at room temperature (RT) for 4 min, or incubated with methanol at –20 °C for 10 min. Cells were incubated with 10 μg/mL of primary antibody (diluted in 0.2% gelatin in TBS) at RT for 2 h. After washes in TBS, coverslips were incubated with FITC- or TRITC- or Cy5-conjugated donkey-anti-mouse or donkey-anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories) at RT for 1 h. DNA was stained with Hoechst 33342 (1:10,000; Molecular Probes, Interchim) at RT for 10 min. After mounting with ProLong Gold Antifade reagent (Molecular Probes), images were acquired with a Zeiss Imager M2 microscope with the Apotome system and a Plan Apochromat 40×/1.3 DIC (oil) controlled by the ZEN software (Carl Zeiss Microscopy GmbH), or a Leica DMRA2 microscope (Rueil-Malmaison, France) equipped with an oil immersion ×100/1.4 apochromatic objective and a 12-bit Coolsnap FX CCD camera (Princeton Instruments, Roper Scientific, Evry, France) controlled by the MetaMorph software (Universal Imaging, Roper Scientific). Images were treated with ImageJ (https://imagej.nih.gov/ij/) or Adobe Photoshop (Adobe Sytems, Inc., San Jose, CA).

Stereological probes were applied using a Zeiss Imager M2 microscope (Carl Zeiss, Jena, Germany) equipped with StereoInvestigator software (v2019.1.3; MBF Bioscience, VT, USA) according to previously published methods [37 (link), 38 (link)]. Using the optical fractionator probe, Tyrosine Hydroxylase (TH) positive cells were counted in sections 480 μm apart using a grid size of 170 X 100 μm and counting frame size of 75 X 75 μm. Contours were drawn around the region of interest at 10X magnification, and cells were counted under a 63X oil immersion objective. Guard zones were set at 2 μm each at the top and bottom of the section, and the counting frame was lowered at 1-2 μm interludes and each cell in focus was marked. The Gundersen method for calculating the coefficient of error was used to estimate the accuracy of the optical fractionator results. Coefficients obtained were generally less than 0.1.

Stereological probes were applied using a Zeiss Imager M2 microscope (Carl Zeiss, Jena, Germany) equipped with StereoInvestigator software (MBF Bioscience, VT, United States) according to previously published methods (Corenblum et al., 2015 (link); Anandhan et al., 2021 (link)). Using the optical fractionator probe, cells were counted under the ×63 oil immersion objective by a blinded observer. TH+ cells were counted in sections 480 μm apart using a grid size of 170 × 100 μm and counting frame size of 75 × 75 μm. The Gundersen method for calculating the coefficient of error was used to estimate the accuracy of the optical fractionator results. Coefficients obtained were less than 0.1.

Female C57BL/6J mice (6–8 weeks old) were injected intraperitoneally with LPS (10 mg/kg), MCL or DXM as indicated. After 12 h, mice were sacrificed and lung or liver tissues were collected to be fixed with 4% formaldehyde and paraffin-embedded. The tissues were sliced and stained with hematoxylin and eosin (H&E). The histopathological changes were observed using a Zeiss Imager M2 microscope (Carl Zeiss MicroImaging) equipped with an AxioCam HRc CCD camera (Carl Zeiss).

C. neoformans strains were observed under a Zeiss Imager M2 microscope, and all images were acquired by an AxioCam MRm camera and processed with Zen pro software version 3.1 (Carl Zeiss Microscopy, Jena, Germany). For heat shock experiments, the cells grown overnight at 22 °C were prepared for microscopy on a 42 °C pre-heated glass slide and incubated at 42 °C for 15–20 min. For the high calcium and salt shock experiments, the cells were suspended in water containing 1 M NaCl or 100 mM CaCl₂ for 15–20 min. The cells were then immediately examined under the microscope. For timelapse experiments involving heating, the cells were prepared on a pre-warmed slide and examined on the same Zeiss Imager M2 microscope equipped with a heated stage (PE120 Linkam stage, McCrone Microscopes & Accessories). For timelapse experiments testing responses to CaCl₂, the cells were suspended in 1 mM CaCl₂ on a microscope slide and immediately imaged.

Female C57BL/6 J mice (6–8 weeks old) were injected intraperitoneally with MRSA (ST 239) (2 × 10⁸ CFU/mouse), MCL or DXM as indicated. After 12 h, mice were sacrificed and liver and kidney tissues were collected to be fixed with 4% formaldehyde and paraffin-embedded. The tissues were sliced and stained with hematoxylin and eosin (H&E). The histopathological changes were observed using a Zeiss Imager M2 microscope (Carl Zeiss Micro Imaging) equipped with an Axio CamHRc CCD camera (Carl Zeiss).