Alexa 594

At 16 weeks after surgery, the tibial nerve was harvested after carbon dioxide euthanasia. The nerve was treated with 4% paraformaldehyde for a duration of 24 h, followed by slicing the nerve into transverse sections of 12 μm thickness using a frozen slicer. Immunofluorescence staining was conducted using an established protocol with NF200 (MilliporeSigma, Burlington, MA, USA) staining targeted toward monitoring axonal regeneration and S100 (MilliporeSigma, Burlington, MA, USA) staining to detect Schwann cells. In short, sections of nerve tissue were incubated with primary antibodies that targeted NF200 and S100 for an overnight period at 4 °C, followed by rinsing with PBS. Afterwards, secondary antibodies conjugated with Alexa488 (Abcam, Cambridge, UK) and Alexa594 (Abcam, Cambridge, UK) were added and incubated at room temperature for 1 h. DAPI (Beyotime, Shanghai, China) was applied to the nucleus and incubated for 5 min. The tissue sections were observed via a 3D fluorescence imaging system (HOOKE INSTRUMENTS, Changchun, China).

Samples from patients with PDR or donor eyecups were fixed with freshly prepared paraformaldehyde (4%) for 2 h at 4 °C, dehydrated using 30% sucrose solutions for 30 min and sent immediately to OCT compound (Sakura, PA, USA) for frozen sections. Frozen sections were cut at 10 μm thickness at −20 °C and stored at −80 °C until staining. Sections were stained by NLRP3 (1:500, Abcam, ab4207) or IL-1β (1:500, Abcam, ab2105) with CD31 (1:500, Abcam, ab24590). HRECs were fixed with freshly prepared paraformaldehyde (4%) for 30 min at 4°C. Then, cells and frozen tissue sections were washed 10 min with PBS (three times), and blocked with 1% FBS in PBS for 1 h at room temperature. After a 10-min washing with PBS three times, samples were incubated with NLRP3 or IL-1β overnight at 4 °C in a humidified chamber. After a 10-min washing with PBS three times, cells were incubated with corresponding secondary antibodies conjugated with Alexa Fluor 488 (1:500, Abcam) or Alexa 594 (1:500, Abcam) for 1 h at 37 °C in a darkened humidified chamber. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA). Fluorescence was observed using a confocal microscope (Olympus 1X81, Olympus, Tokyo, Japan).