Mosaix

Neovascularization analysis in the Vldlr^−/− mice was performed as described (Stahl et al., 2009 (link), Sun et al., 2015 (link)). The whole mounts of retinas from Vldlr^−/− and wild type were stained with isolectin IB4 and imaged using Zeiss AxioObserver.Z1 microscope with a monochrome digital Zeiss camera AxioCam MRm focusing on the terminal end of lesions on the RPE layer (usually at P16), and individual images were merged to create one whole retinal image using automated merge function (mosaiX; Zeiss) in the software AxioVision 4.6.3.0 (Zeiss). ImageJ (National Institutes of Health, http://imagej.nih.gov/ij/) was used for quantification of subretinal neovascularization lesion number and area in Vldlr^−/− retinas with designed plugins adapted from the method used to measure retinal neovascularization (SWIFT_NV) in the OIR model (Stahl et al., 2009 (link)) which use a user-designated threshold to mark lesion structures that clearly stand out from background fluorescence of normal vessels, and can automatically remove small artifacts by selecting objects with a minimum size of 100 pixels. Other larger artifacts such as occasional cellular debris or retinal periphery with hyperfluorescence can be manually excluded from quantification. Lesion numbers and areas were quantified with researchers masked to the identity of samples.

For live imaging, guts were dissected in 1× PBS and immediately mounted in the antifading agent Citifluor AF1 (Citifluor Ltd.). For immunofluorescence, guts were dissected in PBS, fixed for 20 min in 0.1% Tween 20-PBS (PBT) with 4% paraformaldehyde, rinsed in PBT, and then incubated with primary antibodies (1:500 anti-PH3 [Millipore], 1:500 anti-Prospero [DSHB], and 1:1,000 anti-GFP [Roche]) in PBT plus 1% bovine serum albumin. Primary antibodies were revealed with Alexa 488 or Alexa 594-coupled anti-mouse antibodies (Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Guts were then scanned with an Axioplot imager (Zeiss) and recomposed using the software program MosaiX (Zeiss).