Dako mounting agent

Whole eyes from freshly dissected mice were enucleated and fixed in 2% paraformaldehyde (PFA) for 10 min and then the anterior parts were removed. The posterior eyecups were again fixed in 2% PFA for 1 h at room temperature. Immunofluorescence was performed on frozen sections from the posterior eyecups as explained previously [16 (link),21 (link)]. The sections were incubated with phosphate-buffered saline, containing 5% normal donkey or goat serum, for 30 min and then incubated overnight at 4 °C with primary antibodies for Iba1 (Wako, Japan; Cat# 019-19741) or Ly6G (Biorbyt, St. Louis, MO, USA; Cat# orb322983) diluted to 1:100. The sections were washed with TBS and then incubated at room temperature with respective secondary antibodies with 1 µg/mL DAPI (Thermo Fisher, USA; Cat# 62248) and +/− Alexafluor 488-conjugated phalloidin (1:1000) (Thermo Fisher, USA; Cat# A12379). Sections were again washed with TBS and then mounted using DAKO mounting agent (Agilent, USA, Cat# S3023). Images were acquired by a Zeiss LSM 710 confocal workstation.

Eyes were freshly enucleated and then fixed in 2% paraformaldehyde (PFA) for 10 min followed by the removal of the anterior segment (cornea, lens, and attached iris pigmented epithelium). The resulting posterior eyecups were fixed in 2% PFA for 1 h at room temperature for RPE flat mount preparation [17 (link),21 (link)]. Tissues were removed after the eyecup was quartered into a petaloid structure and the neural retina was carefully removed. The resulting eyecup was further cut radially into eight pieces from the optic nerve head to the periphery. Immunostaining on RPE flatmounts was performed by using Alexafluor 488-conjugated phalloidin (1:1000) (Thermo Fisher, USA; Cat# A12379) with 1 µg/mL DAPI (Thermo Fisher, USA; Cat# 62248) and incubated at room temperature for 1 h [17 (link),21 (link)]. The flatmounts were washed six times with 1× tris-buffered saline (TBS), mounted on cover slips with DAKO mounting agent (Agilent, Santa Clara, CA, USA, Cat# S3023), and then visualized under a confocal microscope (Zeiss LSM710, Oberkochen, Germany) to assess RPE morphological changes [17 (link),21 (link)].