80i epifluorescence microscope

Fibroblasts (passage 2, 1×10⁵ cells/mL) or keratinocytes (passage 2, 4×10⁵ cells/mL) were seeded onto electrospun nanofibers collected on coverslips. After 24-h culture, cells were washed with phosphate buffered saline (PBS) and fixed in 3.7 % formaldehyde/PBS for 5 min, then washed extensively in PBS. For overview of the cell distribution and morphology, fixed culture was stained with 0.1% methylene blue and then examined under the Nikon stereomicroscope (SMZ 1500). For immunostaining, fixed cells were dehydrated with ethanol for 5 min, permeabilized with 0.1% Triton X-100 in PBS, and washed in PBS. Cells were stained with TRITC conjugated phalloidin (Sigma) for 40 min, and then washed with PBS to remove unbound phalloidin conjugate. Then one drop of Vectashield mounting medium with DAPI was dispensed onto the coverslip. Cells were viewed at ex360nm/em460nm (DAPI) and ex540nm/em570nm (TRITC) using the Nikon 80i epifluorescence microscope (Nikon Microphot FXA, Melville, NY).

MDA-MB-231 tumor cells and HUVECs were used as model cells to determine the tumor-targeting capability of HA-modified AuNFs. Respective cells (5 × 10⁵ cells per well) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI-1640 media with 10% fetal bovine serum and 1% streptomycin/penicillin for 24 h. The cells were rinsed with PBS and incubated with HA-4-ATP-AuNFs-DOX (100 μg mL⁻¹) for 4 h. To verify the specificity of HA binding to CD44, a separate study was also performed to incubate the cells with free HA (2 mg mL⁻¹) for 1 h prior to the incubation with HA-4-ATP-AuNFs-DOX. Upon further rinsing with PBS, the culture was fixed with paraformaldehyde solution (4%) and observed with a Nikon 80i epifluorescence microscope.
To identify intracellular distribution of AuNFs in tumor cells, MDA-MB-231 cells were seeded into 6-well plates (1 × 10⁶ cells per well) and incubated with HA-4-ATP-AuNFs (100 μg mL⁻¹) for 4 h. Upon rinse with PBS to remove excessive nanoparticles, the cells were trypsinized, washed with PBS, and centrifuged to obtain cell pellets. The pellets were fixed with 2.5% glutaraldehyde for 24 h. Afterward, the pellets were postfixed with osmium tetroxide for 1 h and then rinsed with distilled water. After dehydration in a series of graded ethanol, the pellets were embedded in epoxy for 24 h, and then cut into thin sections (60 nm) for TEM examination.

Optic nerves from different experiments (sections and whole mount) were imaged using a spinning disc confocal microscope (Zeiss) with Velocity software (Perkin Elmer). 20x objective was used to image stacks of all the optic nerves with 20% of overlap between each image. The motorized stage enables the automation of the imaging procedure. All the images were then stitched together using the Velocity software and z-projected to maximal intensity for quantification. Whole mount retinas were imaged with Nikon 80i epifluorescence microscope with 20x objective. At least 8 random fields were imaged per retina.