Fv1200 scanning confocal microscope

Corneal opacity was quantitatively assessed as previously described [29 (link)] with tissue assayed for absorbance at 500 nm using a FLUOstar Omega plate reader (BMG Labtech, Offenburg, Germany) as previously described [59 (link)]. Following the measurement of cornea opacity, the corneas were fixed in a 4% solution of paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min and washed in PBS containing 1% Triton X-100 (Sigma-Aldrich) [29 (link)]. The tissue was then blocked overnight in 10% donkey serum (Abcam, Boston, MA, USA) and labeled for blood and lymphatic vessels as previously described [42 (link)]. Image acquisition was obtained using an Olympus FV1200 scanning confocal microscope in sequential scanning channel mode (Center Valley, PA, USA). The total area positive for blood and lymphatic vessels per field of view (4 quadrants/cornea) was quantified using Metamorph software (Molecular Devices Inc., San Jose, CA, USA).

Nanoformulations were labeled with Coumarin 6 (C6) to measure the effect of trypsin on cell uptake and internalization. 4T1 breast cancer cells were seeded in 12-well plate at a density of 2 × 10⁵ cells/well and incubated for 24 h. After treatment with trypsin for 2 min, cells were incubated with C6 nanoformulations (2 µg/mL C6) for 2 h and collected. Cells were transferred to climbing slices, fixed after 24 h’s recovery, and stained by DAPI. The internalizations of C6 nanoformulations were observed through an FV1200 scanning confocal microscope (Olympus, Japan). Cell samples for flow cytometry were washed, resuspended in phosphate-buffered saline (PBS), and analyzed through FACSVerse™ (BD, USA).
To investigate trypsin’s effect on cell membranes, 4T1 cells were collected after treatment with trypsin for 2 min and 30 min. Cell membranes and nucleus were stained with DiD and DAPI for visualization using confocal microscopy. Meanwhile, cells were treated with 0.5% trypsin for 30 min, washed with PBS, and fixed by electron microscope fixative at 4 ℃ followed by SEM observation.

Images were acquired with an FV1200 scanning confocal microscope (Olympus, Tokyo, Japan) equipped with diode lasers (405, 473, 559 and 635 nm) and analyzed with ImageJ (NIH, Bethesda, MD, USA). For the images of whole slice cultures, Z-series images were acquired with a 0.40 NA 10× objective at a voxel size of 1.242-1.242-4.51 μm (x-y-z). For the analysis of colocalization of the Human Nuclei and HuB or Prox1, Z-series images (40 optical sections) were acquired with a 0.75 NA 20× objective at a voxel size of 0.621-0.621-1.16 μm (x-y-z). For the analysis of colocalization of the Human Nuclei and GFAP, Z-series images (60 optical sections) were acquired with a 0.95 NA 40× objective at a voxel size of 0.3105-0.3105-0.58 μm (x-y-z). GFP-labeled cells were traced with Simple Neurite Tracer (Longair et al., 2011 (link)) in Fiji (Schindelin et al., 2012 (link)). Sholl analysis was performed with Simple Neurite Tracer with the cell body chosen as the center point. The data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. Student’s t test, Tukey’s test or a repeated measure ANOVA test was performed. In all experiments, the samples and cells were analyzed in a double-blind manner.