Dmi3000 inverted microscope

To view overall structure, a subset of oocytes was fixed for 3 days in 3% glutaraldehyde in 0.1 M phosphate buffer, post-fixed for 1h with 1% OsO₄ in 0.1 M phosphate buffer, dehydrated in ascending ethanol series, and embedded in Spurr’s resin. Sections (1 μm) were cut with a Leica EM UC6 ultramicrotome, then mounted and stained with methylene blue and toluidine blue. All light microscopy was conducted using a Leica DMI3000 inverted microscope.

As the trans-azoTAB:PAA microparticles tended to wet the surface of untreated glass coverslips, poly(ethylene glycol) (PEG)-functionalized glass substrates were used for optical microscopy imaging. Optical microscopy was performed on a Leica DMI3000 inverted microscope using a ×63 oil immersion lens, 1.4 NA. Polarized light images were acquired on the same microscope equipped with polarisers. Approximately 2.5 μL of the microparticle suspension were mounted into PEG-functionalized capillary glass slides and observed after 5 minutes of equilibration time. Images of the microparticles were recorded on fields of view containing groups of 20–100 particles. Two to three fields of view were recorded for each experiment. Experiments were repeated at least three times. Images were analysed using ImageJ. The circularity (Ψ) of the particles was estimated from Ψ = d_min/d_max where d_min and d_max are the perimeters of the minimum and maximum circumscribed circles associated with each particle48 .

MCF-7 breast cancer cells (American Type Culture Collection) were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone), supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin (100 µg mL⁻¹, Gibco), and streptomycin (100 µg mL⁻¹, Gibco) in a humidified environment (37 °C, 5% CO₂). Before treating with nanoparticles, the cells were seeded onto cover glass in a six-well plate with DMEM. The prepared CdSe/ZnS@FA QDs and GNR@CdSe/ZnS@FA nanoparticles were then diluted with PBS buffer (pH 7.2) solution to a concentration of 500 μg/mL. Next, the cells were treated with the CdSe/ZnS@FA QDs and GNR@CdSe/ZnS@FA nanoparticles for 4 h. After 4 h of incubation, the treated cells were washed with PBS buffer three times. A Leica DMI 3000 inverted microscope with a 10× lens was used for the cell imaging study, and the excitation and emission wavelengths were 532 nm and 630 nm, respectively.

KRC and SVEC4–10 cells were cultured in 3D as described [32 (link)]. Briefly, KRC PCCs were transduced with an eGFP lentiviral construct (Clontech), and SVEC4–10 ECs were labeled using the PKH26 red fluorescent cell linker kit per the manufacturer's recommendations (Sigma-Aldrich). 3,000 PCCs or 6,000 ECs were cultured alone or together in 3% matrigel. Two days after plating, and every two days thereafter, cells were treated with control media, or media with ruxolitinib [100 nM]. The final concentration of DMSO in all experiments was 0.05%. Images were acquired with a Leica DMI3000 inverted microscope outfitted with a DFC300 FX camera. Fluorescence intensity was determined on three independent experiments plated in duplicate using Image Pro Plus v.7 (Media Cybernetics).

Scratch wound-healing assays were performed in 24-well tissue culture plates. The cells were seeded at a density of 1X10⁵ cells/well. Scratches were made using 1 mL sterile pipette tips and the wells were washed twice with medium. Cells were allowed to grow for additional 24 h or 48 h in the presence or absence of grifolin. Images were taken under a Leica DMI3000 inverted microscope. Gap distance of the wound (μm) was measured using LAS V3.8 software. The alteration of cell migrated distance was calculated using the formula of (B-A), Where, A denotes the gap distance at 24 h or 48 h, and B for gap width at 0 h.

To examine patency, freshly dissected ovarioles were stained with 1% Evans Blue and processed as described in Pascual et al. [43] . To view overall structure, a subset of oocytes were fixed for 3d in 3% glutaraldehyde in 0.1 M phosphate buffer, post fixed for 1 hour with 1% OsO₄ in 0.1 M phosphate buffer, dehydrated in an ascending ethanol series, and embedded in Spurr's resin. Sections (1 µm) were cut with a Leica EM UC6 ultramicrotome, then mounted and stained with methylene blue and toluidine blue. All light microscopy was performed using a Leica DMI3000 inverted microscope.
For fluorescence imaging, ovarioles were fixed and stained with phalloidin–TRITC (cytoskeleton; Sigma) and DAPI (nuclei; Sigma) as per Cruz et al. [44] (link), then examined with a Leica TCS SP8 confocal microscope.
Transverse 100 nm ultrathin sections were taken near the midpoint between the anterior and posterior ends of the basal oocytes embedded in Spurr's resin above. Ultrathin sections were mounted and stained according to [45] (link) and follicle cell ultrastructure was then imaged using a Hitachi HT-7700 transmission electron microscope and AMT Image Capture Engine Software. All images were post processed in Adobe Photoshop.