Nb300 223

To detect the expressions of specific markers in Müller cells (Vimentin) and retinal progenitors (CHX10), Müller cells were cultured in six-well plates which covered with coverslips for 24 h and subsequently starved in serum-free DMEM for 8 h. Following treatment with PKH67 labeled-hESEVs, MVs, EXOs, sh-NC-MVs, sh-HSP90-MVs, pcDNA3.1-MVs, or pcDNA3.1-HSP90-MVs, the Müller cells were washed thrice with PBS, followed by 20 min of fixation in 4% formaldehyde solution (Thermo Fisher Scientific, MA, USA) at room temperature and 10 min of permeation with PBS containing 0.1% triton-X-100. Then, cells were washed with PBS and blocked with sheep serum (Gibco, NY, USA) (1:50) for 30 min. Following 5 min of PBS washing thrice, cells were subjected to the primary antibody against Vimentin (NB300-223, 1:5000) or CHX10 (NBP1-84476, 1:1000) (Novus, St. Louis, MO, USA) overnight at 4 °C for incubation. Cells were washed with PBS thrice and incubated with Texas Red-labeled sheep anti-mouse IgG (ab6787, 1:1000, Abcam, USA) at room temperature for 2 h. The nuclei of Müller cells were stained with DAPI (4′,6′-diamidino-2-phenylindole; Vector Laboratories, Inc., Burlingame, CA) at room temperature for 5 min prior to capture the fluorescence images under immunofluorescence microscopy (Olympus IX71, Tokyo, Japan).

Following PBS washing for 3 × 5 min, the retinal sections were incubated with 0.1% Triton for 10 min, subjected to normal rabbit serum (Invitrogen, CA, USA) to block non-specific binding sites, and then incubated at 37 °C for 30 min to discard serum. Then, primary antibody against Vimentin (NB300-223, 1:5000) or CHX10 (NBP1-84476, 1:1000) (Novus, St. Louis, MO, USA) was incubated with sections overnight in a humidified box at 4 °C (the NC group was treated with 0.01 mmol/L of PBS). Then, sections were washed thrice with PBS for 5 min, followed by incubation with the secondary antibody labeled with FITC fluorescence (ab6662, 1:1000, Abcam, USA) or Texas Red (ab6787, 1:1000, Abcam, USA) in a humidified box at 37 °C for 40 min. Following 5 min of PBS rinsing thrice, sections were sealed with water-soluble resin. Pictures were captured with a fluorescence microscope (Olympus IX71, Tokyo, Japan). Analyses of images were performed by using Fiji [16 (link)] and associated plugins as previously described [17 (link)].