Fig. 8

Ventromedial hypothalamic neurons with inclusions from Gfa2-CGG99 mice do not express eGFP by qPCR analysis. A1–3: (a1) Neurons in ventromedial hypothalamus (VMH) containing ubiquitin positive inclusions were visualized in 14 μm cryosections of brain by immunofluorescent labeling (green immunofluorescence). (a2) Same section with DAPI staining. (A3) Neurons with inclusions were collected by laser capture microdissection (LCM). b Total RNA was extracted and amplified from LCM neurons and qPCR analysis was performed to detect expression of eGFP. VMH neurons from Gfa2-CGG99 mice with inclusions did not show detectable expression of eGFP (GFA2 LCM Neurons). In contrast, eGFP expression was readily detected in large LCM samples from cerebral cortex that included astrocytes with eGFP expression (GFA2 Cortex). H₂O was a water control. As an additional tissue control, single neurons from the amygdala of a CGG KI mouse (168 CGG repeats) were isolated by LCM and analyzed for eGFP expression (CGG KI Amygdala), and as expected since these mice do not carry the eGFP reporter gene, no expression was detected. Red dashed line shows the detection threshold for qPCR analysis. c Analysis of qPCR samples by gel electrophoresis confirmed amplification of eGFP in cortical samples containing eGFP positive astrocytes, but not in samples of LCM-isolated hypothalamic neurons. L, DNA ladder; 1, CGG KI amygdala; 2, GFA2 LCM neurons; 3, GFA2 cortex; 4, water; 5, eGFP and/Actin positive plasmid controls. Scale bar = 50 μm