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. 2015 Feb 4;24(10):2808–2825. doi: 10.1093/hmg/ddv042

Figure 5.

Figure 5.

Response of cultured GCPs to SHH and exogenous cholesterol. (A) Immunofluorescent staining for BrdU (red) and neuronal marker TuJI (green), with DAPI (blue) counterstaining of Nsdhlflx5/Y and NsdhlΔ5/Y GCPs that were cultured for 48 h with either 1 μg/ml recombinant SHH alone or SHH plus 15 μg/ml cholesterol. BrdU was added to the samples 4 h before fixing. Note that there are fewer and thinner neurite extensions in the mutant NsdhlΔ5/Y cells than in the Nsdhlflx5/Y controls. Scale bars: 100 μm. (B) Four aliquots of isolated GCPs from individual Nsdhlflx5/Y and NsdhlΔ5/Y P4 cerebella were cultured for 48 h in either serum free medium (SFM) alone, SFM with 15 μg/ml cholesterol (chol), SFM with 1 μg/ml recombinant SHH, or SFM with 15 μg/ml cholesterol and 1 μg/ml SHH. BrdU was added to the cultures 4 h before fixing the cells. Cells were immunostained for BrdU and counterstained with DAPI. Values are the percentage of BrdU-labeled cells from the total (DAPI-stained) number of cells. Each bar represents the mean ± SEM of results from three independent experiments.