After enucleation, eye cups and retina were carefully removed. RPE flatmounts were fixed in 2% paraformaldehyde, stained with rabbit polyclonal antibodies against mouse ZO-1 (1:100; Invitrogen, Carlsbad, CA, USA), and visualized with Invitrogen Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, USA). For immunofluorescence staining for Cre expression, fresh, unfixed mouse eyes were embedded in optimal cutting temperature compound (Thermo Fisher Scientific), frozen in isopentane precooled by liquid nitrogen, and then cryosectioned. Using a Nikon A1R confocal microscope system (Nikon, Tokyo, Japan), flatmounts were imaged and assessed by a blinded operator. Degeneration in RPE flatmounts was defined using an established protocol.20 (link)
Protocol detail
Immunostaining of Retinal Pigment Epithelium
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Alexa 594
Antibodies
Confocal microscope
Eyes
Frozen
Immunofluorescence
Isopentane
Mouse
Nitrogen
Paraformaldehyde
Rabbit
Retina
Corresponding Organization : University of Virginia
Other organizations : Shanghai Jiao Tong University, Shanghai First People's Hospital, Aravind Eye Hospital, Universidade Federal de São Paulo, University of Tsukuba, Doheny Eye Institute, University of California, Los Angeles
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Variable analysis
independent variables
- Enucleation of eyes
- Removal of eye cups and retina
- RPE flatmounts were fixed in 2% paraformaldehyde
- RPE flatmounts were stained with rabbit polyclonal antibodies against mouse ZO-1 (1:100; Invitrogen, Carlsbad, CA, USA)
- RPE flatmounts were visualized with Invitrogen Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, USA)
- Fresh, unfixed mouse eyes were embedded in optimal cutting temperature compound (Thermo Fisher Scientific), frozen in isopentane precooled by liquid nitrogen, and then cryosectioned
- Degeneration in RPE flatmounts
- Blinded operator for imaging and assessment of RPE flatmounts
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