Fv3000 microscope

RH30 and RD cells were fixed after 8 and 24 hours of either vehicle, each drug alone, or combination treatment in 4% paraformaldehyde (PFA)/PBS for 15 min at RT, permeabilized in 0.2% Triton X-100/PBS for 5 min at RT and incubated with either rabbit phospho-MET (Tyr1234/1235) (#3077 from Cell Signaling Technology, (CST) Danvers, MA, USA) and rabbit MET (sc-10 from Santa Cruz Biotechnology, Inc., Heidelberg, Germany) in 1% BSA/PBS. Alexa-488 goat α-rabbit (Invitrogen, Carlsbad, CA, USA) was used as secondary antibody. Cells were counterstained with DAPI and imaged using the Olympus microscope FV3000 with Olympus FV315S-SW image acquisition software. Images were processed by Adobe Photoshop. The intensity average of phospo-MET and MET fluorescences were calculated using FV315S-SW software, from cytometric measurements relative to total cell area in 5 digital images randomly selected and analyzed for each immunostained cellular sample. From 80 to 100 cells were counted for each sample analyzed.

Nucleic acid damage such as DNA fragmentation and condensation due to treatment with the antifungal lipopeptide AF₄ was assessed using DAPI (HiMedia, India), a nucleic acid stain. Cell suspensions of C. tropicalis and C. auris were treated with antifungal lipopeptide at 2-, 4-, and 8-mg/L concentrations to observe dose-dependent effects on the nucleic acid content of the cells. Cells (~5 × 10⁶ CFU/mL) were incubated with AF₄ for 18 h in RPMI 1640 at 37°C under shaking conditions. Untreated cells were used to observe the intact nuclei. Cells harvested at 10,000 rpm post-treatment were stained with 1 μg/mL DAPI for 20 min at 37°C (21 (link), 23 (link)). Cells were imaged using an Olympus FV3000 microscope (CSI Facility, BITS Pilani Goa Campus).

Cells forming a 50–70% monolayer on coverslips were pretreated with the 5 mM methyl-β-cyclodextrin (Sigma-Aldrich, Munich, Germany) for 30, 60 or 120 min. Cells were washed three times with PBS solution at each staining step. The preparations were fixed with 3.7% formaldehyde solution (Sigma-Aldrich, Munich, Germany) for 10 min. For labelling the lipid rafts, cells were stained with cholera toxin subunit B conjugated with FITC (Sigma-Aldrich, Munich, Germany) for 15 min at 4 °C. To visualize the actin cytoskeleton, the cells were incubated with 0.1% Triton X-100 for 5 min and stained with rhodamine-phalloidin for 15 min at 37 °C. To visualize the DNA of the epithelial cells, the cells were stained with DAPI for 5 min. The preparations were analyzed using an Olympus FV3000 microscope (Olympus, Tokyo, Japan) with a system of lasers with wavelengths of 405 (blue fluorescence), 488 (green fluorescence) and 561 nm (red fluorescence).

The surface morphologies of the colloidosomes were observed by scanning electron microscopy (SEM, JEOL Co., Tokyo, Japan). Bare and modified silica particles in pellet form were generated following the addition of spectroscopic grade KBr. The Fourier transform infrared spectroscopy (FTIR) spectra were recorded using a 1615 FTIR spectrometer (PerkinElmer, Waltham, MA, USA). The particle size distribution of the Janus particles was measured by a laser diffraction method (Mastersizer 2000, Malvern Instruments Ltd., Malvern, UK). The fluorescent amphiphilic nanoparticles and foams were optically analyzed using a FV3000 microscope (Olympus, Tokyo, Japan) with an electron CCD camera.

Cells were imaged using PLAPON 60x/1.42 oil objective of Olympus FV3000 microscope. Images were taken with intervals of 1.1 seconds.

Mice anesthetized with isoflurane were transcardially perfused with 0.1 M PBS and 4% paraformaldehyde in PBS. Brains were removed and post-fixed in 4% paraformaldehyde at 4 °C overnight, dehydrated in 20%, and then 30% sucrose until they sank. Coronal sections (35 μm) were sliced on a cryostat (Leica CM1950). For immunofluorescence, sections were washed with PBS three times (5 min each) and were incubated with blocking buffer (0.3% Triton X-100, 10% goat serum in PBS) for 1 h at room temperature, then they were incubated (12–24 h at 4 °C) with the primary antibodies anti-TPH2 (1:500, rabbit, Abcam, Cat# ab111828), anti-VGluT3 (1:500, rabbit, Synaptic Systems, Cat# 135203), anti-GABA (1:500, rabbit, Sigma, Cat# A2052), anti-TH (1:1000, rabbit, Emd Millipore, Cat# AB152). After rinsing with PBS, the sections were incubated with the corresponding fluorophore-conjugated secondary antibodies (Goat anti-rabbit Alexa Fluor 488, Goat anti-rabbit Alexa Fluor 594 and Goat anti-rabbit Alexa Fluor 647, 1:500, Jackson ImmunoResearch Labs, Cat# 111-545-003, Cat# 111-585-003 and Cat# 111-605-003) for 2 h at room temperature. Sections were then washed three times with PBS, stained with DAPI, and coverslipped with fluorescent mounting medium. Confocal images were captured on an Olympus FV3000 microscope and analyzed with ImageJ software.