Lsm 780 confocal microscope

MSCs were grown on 24-well plates and cultured with conditioned medium as indicated. After 48 h, the cells were fixed in 3.7% formaldehyde for 20 min, permeabilized by 0.2% Triton X-100 for 10 min. After treatment with 10% goat serum for 30 min, primary antibodies and species-specific secondary antibodies conjugated with either Alexa Fluor 594 or Alexa Fluor 488, and nuclei were counter-stained with DAPI (4′,6-diamidino-2-phenylindole) for 10 min. Images were acquired using an LSM780 confocal microscope (Zeiss).
For direct immunofluorescence staining in ear sections, 5 μm cryo-sections were fixed in 4% paraformaldehyde (PFA), blocked in 5% FBS plus 5% rat serum and incubated with the following antibodies: Alexa Fluor® 594 anti-mouse CD8a anti-body (Biolegend), nuclei were counterstained using DAPI. Images were acquired using an LSM780 confocal microscope (Zeiss). CD8⁺T cells in the ear epidermis were quantified in 3–5 images per specimen using ImageJ software.

To examine the morphology of the pyroptotic cells, the cells were seeded in 6-well plates at ~40%-60% confluency and then treated with different drugs. Phase contrast cell images were captured under a Nikon TE2000 microscope.
For confocal microscopy, the cells were washed with DMEM and fixed in 4% paraformaldehyde, followed by blockade with blocking buffer (3% BSA and 0.2% Triton X-100). The cells were incubated with the appropriate primary antibody overnight at 4 °C, washed with washing buffer (0.2% BSA and 0.05% Triton X-100) and incubated with FITC- or Texas Red-conjugated secondary antibodies (Life Technologies) for another 1 h at 37 °C in the dark. The cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, 50 μg/mL) for 5 min to indicate the nuclei. Images were captured under a Zeiss LSM 780 confocal microscope.
For nuclear staining, the cell nuclei were stained with Hoechst33342 in the dark for 10 min at room temperature and the cells were imaged using a Zeiss LSM 780 confocal microscope.

To analyze possible changes in cell membranes caused by limonene formulation, we stained the cell membrane with a commonly used membrane-selective fluorescent dye FM4-64 (Fischer-Parton et al., 2000 (link)). Simply, the treated mycelia were immersed into FM4-64 solution (7.5 μM) for 1 min at room temperature, and immediately observed the stained mycelia under a Zeiss LSM780 confocal microscope (Gottingen, Niedersachsen, Germany).
The vacuole lumenal marker 7-amino-4-chloromethylcoumarin (CMAC) was used to evaluate the influence of limonene formulation on vacuoles. The mycelia of each treatment were stained with 100 μM CMAC solution at room temperature for 30 min, and washed by sterile water for three times. Finally, the stained mycelia were subjected to microscopic analysis using Zeiss LSM780 confocal microscope.
To investigate the effect of limonene formulation on the formation of lipid droplets, the hyphae of the treatment were stained with Nile Red, which is the most commonly used fluorescent dye to quantify neutral lipid content (Kimura et al., 2004 (link)). Each sample was stained with Nile Red staining solution, which containing 20 mg/ml polyvinylpyrrolidone and 2.5 mg/ml Nile Red Oxazone (Sigma-Aldrich, HY-D0718) in 50 mM Tris-maleate buffer (pH 7.5). After staining for 2 min, the lipid droplets were observed under Zeiss LSM780 confocal microscope.

To evaluate the contractile function of 3D hOCMTs, live image sequences were acquired with Zeiss LSM 780 Confocal microscope using transmitted light mode (37 °C, 5% CO₂), as equivalent to 60 fps for 15 s; where the image sequences were later turned into .avi files with ImageJ. The beating rates were counted and calculated manually from the video analysis, and representative contraction graphs were derived with the open-source video analysis software MUSCLEMOTION^{86 (link),87} per providers’ instructions.

Five-day-old thalli were used for observation. For BODIPY staining, thalli were incubated in 200 nM BODIPY 493/503, dissolved in water for 30 min, washed twice in water, and used for microscopic observation. For β-estradiol treatment, gemmae were grown on a 1/2 × Gamborg’s B5 medium plate for 3 days, then soaked in a liquid medium containing 20 μM β-estradiol or 0.1% (v/v) DMSO for 48 h. The samples were mounted in a 1/2 × Gamborg’s B5 liquid medium and observed using an LSM780 confocal microscope (Carl Zeiss) equipped with an oil immersion lens (63 ×, numerical aperture = 1.4) and lambda and Airyscan detectors. For spectral imaging, the samples were excited at 488 nm (Argon 488) and 561 nm (DPSS 561–10), and emissions between 482 and 659 nm were collected. For high-resolution imaging using the Airyscan detector, samples were excited at 488 and 561 nm, and the emission was separated using a BP495-550 + BP570-630 filter. The images were acquired using line scanning. Spectral unmixing and Airy processing of the obtained images were performed using ZEN2.3 SP1 software (Carl Zeiss). We calculated the circularity of the oil bodies and analyzed colocalization using ImageJ and the Coloc_2 plugin in ImageJ Fiji (Schindelin et al., 2012 (link)), respectively.

The pHAM1::YPET-N7 and pHAM2::YPET-N7 reporter lines (Ler background) have been described previously.^{18 (link)} Plants were grown on soil (Levington F2), on a mixture of half soil and half sand, and watered with or without 1/1/1 fertiliser (Vitafeed Standard) and placed in a constant light room (24 h light, 22 °C, intensity: 160 μmol m⁻² s⁻¹) until bolting stage. Imaging was performed as follows: the main inflorescence meristem was cut, dissected under a binocular stereoscopic microscope to remove all the flowers down to stage 3 (as defined in ref.^{27 (link)}) and transferred to a box containing an apex culture medium as described in ref.^{28 (link)} Meristems were imaged in water using a 20× long-distance water objective mounted on a LSM780 confocal microscope (Zeiss, Germany). Z-stacks of 2 μm spacing were taken. Z-projections (Sum slices) and orthogonal sections were performed using the ImageJ software (https://fiji.sc/).
The plant count for every conditions was:
pHAM1::YPET-N7:

Soil: 17 plants

Soil + fertiliser: 11 plants

Soil + sand: 20 plants

pHAM2::YPET-N7:

Soil: 16 plants

Soil + fertiliser: 12 plants

Soil + sand: 16 plants