Preparation of Retinal Tissue Sections

After the culturing of organotypic retinal explants with CD or saline, the cultures were fixed in a 4% paraformaldehyde/PBS solution for at least 45 min. The explants were cryoprotected by introducing an incremental amount of sucrose to the well plate, i.e., 10% sucrose, 20% sucrose, and 30% sucrose for 10, 20, and 30 min (at RT), respectively. Afterwards, the area of the retina culture attached to the Transwell membrane was cut out with fine scissors, and the membrane piece was submerged in an embedding medium (Tissue-Tek O.C.T. Compound, Sakura Finetek Europe, Netherlands), followed by snap freezing in liquid nitrogen. The frozen specimens were sectioned on an NX50 cryostat (ThermoFisher, Waltham, MA) to produce 14 µm thick sections on Superfrost Plus object

slides (R. Langenbrinck, Emmendingen, Germany) used for direct imaging or further staining.

Assessing Cell Death in Retinal Sections Using the TUNEL Assay

A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to stain the nuclei with damaged (nick-end) DNA [56]. This was done to quantify the number of dying cells in retinal cultures after CD or saline was applied to assess the cytotoxicity of the CDs. Firstly, microscopy slides with retinal sections were rehydrated with PBS. A proteinase K solution preheated to 37 ◦C (0.21 µg/mL, Tris-buffered saline (TBS) (Sigma-Aldrich, Saint Louis, MO, USA) was added and incubated for 5 min at 37 ◦C. After washing with TBS, a solution of ethanol/acetic acid was added and incubated for 5 min before washing. A blocking solution consisting of 1% bovine serum albumin, 10% normal goat serum, 3% Triton-X (Sigma-Aldrich, Saint Louis, MO, USA), and 2.5% fish gelatin in PBS was incubated on the sections (1 h, RT). A TUNEL reaction solution consisting of the enzyme solution and labeling solution from the In Situ Cell Death Detection Kit (TMR red, Product No. 12156792910, Sigma Aldrich) was prepared at a 1:9 ratio, diluted in blocking solution (1:1), and incubated with the slides at 37 ◦C for 1 h. The slides were washed with PBS, and a mounting medium with DAPI (Vectashield, Vector Laboratories, USA) was added. Samples were kept at 2–8 ◦C for at least 30 min before imaging with fluorescent microscopy (Axio Imager Z2 ApoTome, Carl Zeiss Microscopy GmbH), using a CCD camera with a 20× objective. Ex./Em. of 548/561 nm was used to detect TUNEL labeling at random locations of the section. Image acquisition was conducted by recording z-stacks, each with 10 images 1 µm apart. Values such as the exposure time for the red (TUNEL) channel, binning, and brightness/contrast of each image were kept consistent. To quantify the number of dying cells, in either the inner or outer nuclear layer in the tissue section, the following equation was used:

TUNEL positive cells (%) = #TUNEL positive nuclei Area of layer / Average area of nuclei·100%. (1)

To analyze the statistical significance within the dataset, one-way ANOVA with Tukey's multiple comparisons test (α = 0.05) was performed using GraphPad Prism 8.

Determining the Retinal Uptake of Fluorescently-Labeled β-Cyclodextrin

Fluorescently-labeled CD (FITC-HPβCD, RBITC-HPβCD, and FITC-RMβCD) was added to organotypic retinal explant cultures, as described previously. Slides with retinal tissue sections were rehydrated in PBS for 10 min before mounting medium with DAPI (Vectashield, Vector Laboratories, USA) was added. Images were recorded with fluorescent microscopy, as described above. A green (Ex./Em. 493/513) and red (Ex./Em. 558/575) channel were used to detect the fluorescein or rhodamine labeling, respectively. Image acquisition was conducted by recording z-stacks, each with 14 images 1 µm apart. Values such as the exposure time for the green and red channel, binning, and brightness/contrast of each image were kept consistent. Sections from the saline-treated retinas were imaged using the same parameters to determine the level of green and red auto-fluorescence from the cultures. Alternatively, tile pictures showing the entire retinal section were recorded by stitching together adjacent projected z-stacks in the image acquisition software (ZEN 2.6, Carl Zeiss Microscopy GmbH).
