Figure 5

Utilizing traditional techniques in combination with scRNAseq data to understand the response of immune cell populations during acute photoreceptor degeneration. (a) Relative expression of H2-Aa, Ccr2, and Ccl2 across clusters. (b) Complete gating strategy for identifying alive, single, captured CD11b⁺CD45^high cells. The gating strategy is shown here on the degenerating sample. (c) MHCII and CCR2 protein expression on CD11b⁺CD45^high cells gated for scRNAseq. There was an increase in MHCII^high events (blue box) and CCR2 (red box) in degenerating tissue. Histograms on the edge of plots show the distribution of events across fluorescence intensities of MHCII and CCR2. (d,e) During degeneration a subset of macrophage-like CD11b⁺ cells express MHCII. No MHCII expression is regularly detected in control Arr1^−/− retinas. In retinal sections, (d) MHCII⁺ cells can be seen in multiple retinal layers. Retinal flatmounts (e) clearly reveal that some, but not all, macrophage-like cells express MHCII during degenerationg, and there are no clear morphological distinctions between MHCII⁺ and MHCII⁻ macrophage-like cells. Additionally, during degeneration proliferating macrophage-like CD11b⁺ cells can be identified in the retina by nuclear Ki67 staining. Ki67⁺ nuclei are not observed in dark-reared control retinas, likely due to the very low levels of microglia turnorver at rest. Both MHCII⁺ (magenta, due to overlap of red and blue) and MHCII⁻ (red) CD11b⁺ cells with Ki67⁺ nuclei (green) can be observed in degenerating retinas. Scale bars in retinal sections indicate 25 µm; scale bars in flat mounts indicate 50 µm. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer.