Extended Data Fig. 2: Identification of astrocyte γC3 gene expression in V1.
a, Low-magnification image illustrating γC3 expression in astrocytes across both upper (L2/3) and lower (L5/6) cortical layers. γC3 expression is also expressed in other cell types in these regions. Scale bar, 40 µm. Representative images are from experiments quantified in (b), which were repeated independently in three mice. b, Astrocyte cell areas were segmented based on the expression of the astrocyte-specific marker Slc1a3, detected using an RNAscope in situ hybridization probe. γC3 expression was detected using an isoform-specific RNA probe. Solid outlines indicate the cell boundaries of identified single astrocytes. γC3 RNA puncta per cell were quantified within Slc1a3-positive regions in layers 2/3 and 5/6 of V1 in WT mice at P21. N = 280 cells from three mice. Statistical comparisons were performed using the two-sided Wilcoxon rank-sum test, with nested analysis treating the animal as the unit of analysis. Error bars represent the standard error of the mean (s.e.m.). The probe specificity to γC3 is supported by two observations. First, each of the multiple probes (referred to as Z probes) in the probe set is assessed computationally for cross-reactivity with non-cognate sequences in the transcriptome. Second, signal amplification requires adjacent Z probes to bind simultaneously, which contributes additional stringency in signal detection. Scale bar, 10 µm.
