Our data validated the feasibility of astrocyte-specific sampling to detect cell-specific differences in protein expression in experimental glaucoma. We initially aimed to verify our enriched astrocyte samples by Western blot analysis using specific antibodies to different retinal cell markers. Similarly isolated RGC samples were verified previously based on retrograde fluorescence labeling,
15 cell morphology and immunolabeling in culture,
15 RT-PCR,
20 and Western blot analysis of specific markers.
18 However, to provide complementary information, we re-examined RGC samples, and also retinal protein samples obtained after depletion of astrocytes and RGCs, along with the analysis of astrocyte samples (
Fig. 1A). Western blots verified GFAP and ASTRO1 expression in selected astrocytes. However, neuronal markers, including NeuN and Thy-1.1, were not detectable in these samples. Despite faint immunoreactivity for GS (a marker for Müller cells) and CRALBP (expressed in Müller cells and retinal pigment epithelium), enriched samples of astrocytes were negative for RPE65, a marker for retinal pigment epithelium. Similar to previous observations, RGC samples were positive for NeuN and Thy-1.1
, but negative for ASTRO1, GFAP, GS, CRALBP, or RPE65. The minimum contamination of enriched astrocyte samples with Müller cell proteins (GS and CRALBP expressed only by developing astrocytes
27 ) likely is due to the ASTRO1 antibody used for cell isolation, which is specific for astrocytes but also may bind weakly some Müller cells in the retina. To provide an internal control and complementary information,
Figure 1A also presents Western blots of retinal protein samples obtained after depletion of astrocytes and RGCs. Note that dark immunoreactive bands for GS and CRALBP support Müller cell proteins remaining in these astrocyte/RGC-depleted samples. Altogether, the data presented in
Figure 1A indicate no prominent contamination of enriched astrocyte samples with other retinal cell types and support the high purity of these samples.