Mouse Eye Retinal Flatmount Staining

Whole mouse eyes were collected and fixed in 2% PFA/PBS for 2 h before transferring to PBS and stored in 4 °C for further processing [20 (link)]. For retinal flatmount staining, retinal tissues were permeabilised and blocked in 1% BSA/PBS for 2 h and then incubated in primary antibodies (Biotinylated Griffonia Simplicifolia Lectin I Isolectin B4: 1:50, Vector laboratory, Peterborough, UK; rabbit anti-mouse Collagen IV: 1:50, AbD Serotec®, Kidlington, UK) overnight. After thorough washes, samples were incubated with secondary antibodies (Streptavidin FITC or goat anti-rabbit Alex Fluor 569, all from Invitrogen, UK, 1:200) at room temperature for 2 h. Tissues were flatmounted on glass slides with mounting medium and evaluated under confocal microscope (EZ-C1 confocal system, Nikon UK Limited, Surrey, UK).