Fig. 3: Lipe−/− mice demonstrate increased accumulation of activated subretinal microglia.
a Diagram showing the imaging protocol for RPE flat mounts with each square representing a 20× field (664 µm by 664 µm). Central (C), paracentral (PC), mid-peripheral (MP) and peripheral (P) regions were imaged in each quadrant. This diagram was generated with BioRender.com. b Iba1+ cells were counted in flat mounts from Lipe−/− and Lipe+/+ mice (n = 12 fields per group per region). Lipe−/− mice showed a statistically significant increase in Iba1+ cells in all imaged regions (central, paracentral, mid-peripheral and peripheral) compared to Lipe+/+ mice. Data are presented as Mean ± SEM. Two-tailed Student’s t test; *p < 0.05, **p < 0.01. Analysis was done both including all values (black asterisks), and also excluding outliers (blue ellipses and blue asterisks). c Flat mounts were then co-stained with Iba1 (red) and CD16 (green) and a representative 20× image is shown. d A magnified view of the blue squared area in Fig. 3c is shown, to highlight examples of microglia with typical activation morphology (less extensions and larger cell bodies; shown in white squares). Note that microglia with morphological signs of activation are also co-staining with CD16 and are thus appearing more orange/yellow. These should be compared to those in white circles, which have less of an activation morphology (more extensions) and are not staining for CD16 (they appear more red). Scale bar in panel c =75 µm. e CD16+/Iba1+ ratio obtained from central (n = 8) and paracentral (n = 23) fields. The central fields show a statistically significant increase in the proportion of Iba1+ cells that are also CD16+ when compared to paracentral fields in Lipe−/− flatmounts. Data are presented as Mean ± SEM. Two-tailed Student’s t test. *p < 0.05, **p < 0.01.
