Fig. 5.
The ELISA can be used to screen for molecules involved in glial-mediated regulation of synaptic protein levels during developmental synapse elimination. The ELISA was used to assess synapse elimination during development using the endogenously tagged synaptic protein, Bruchpilot-GFP (Brp-GFP). Gene expression was manipulated in astrocytes using the GMR25H07 driver. (A) An endogenously tagged Brp protein was previously generated using a MiMIC insertion of GFP into the endogenous brp locus. (B) The CNS was dissected from pupae at HE, ∼12 h into metamorphosis. Immunohistochemistry was performed for Brp, and the endogenous Brp-GFP signal was used to evaluate synapses. Images were acquired across the entire CNS and images shown are maximum z projections from two stitched 20x images. (C) GFP+ area within the VNC neuropil was quantified from single z planes (UAS-nls-lacZ n = 15, UAS-EcRDN n = 14, UAS-drpr RNAi n = 14, UAS-ced12 RNAi n = 14). (D) Five dissected CNSs were collected for each sample and GFP levels were assessed by ELISA (UAS-nls-lacZ n = 4, UAS-EcRDN n = 4, UAS-drpr RNAi n = 4, UAS-ced12 RNAi n = 4). Graphs represent mean ± SEM and the dotted line indicates the mean of the control UAS-nls-lacZ genotype. Statistical comparisons were performed using unpaired two-sided t tests between each experimental genotype and the control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, or as indicated. (Scale bar, 50 μm.)