Extended Data Fig. 2: Both AAV5-GfaABC1D-Cx43:TID:HA and biotin are required for astrocyte network detection.
From: Astrocytes connect specific brain regions through plastic networks
a. (also in Fig. 1e) Primary rat astrocytes (GFAP, green) infected with AAV5-GfaABC1D-Cx43:TID:HA and given biotin have punctate staining patterns for HA-tag in about 20% of cells; ~80% of cells are positive for streptavidin. b. Without supplemented biotin, HA-tag is detected but not streptavidin. Without AAV (c) or with neither AAV nor biotin (d), neither HA-tag nor streptavidin are detected. e. Primary rat astrocytes were incubated with a range of MOIs (multiplicity of infection) to estimate the viral load necessary for ~10% of astrocytes to express Cx43-TID. Across each MOI tested, about 80~ of astrocytes were positive for biotinylated fluxed molecules. n = 2 isolations f. 87.7 ± 3.0% of cells from the AAV + /Biotin+ condition (MOI = 1 ×104) were positive for streptavidin; the other 3 conditions contained no detectable streptavidin signal. g. 13.3 ± 0.8% of cells were positive for HA-tag in both AAV+ conditions; neither AAV- condition contained detectable HA-tag immunopositivity. n = 3 isolations. Statistical significance (p values for all comparisons < 0.0001) determined via one-way ANOVA followed by Tukey’s multiple comparisons test (α = 0.05).
