Computational protocol: Synaptic Phospholipid Signaling Modulates Axon Outgrowth via Glutamate-dependent Ca2+-mediated Molecular Pathways

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Protocol publication

[…] Axon outgrowth assays were performed and analyzed as described () using ImageJ. Briefly, EC slices were prepared at postnatal day 0/1 and were embedded in a collagen I matrix on glass slides and covered with nutrition medium. An inhibitor cocktail containing TTX (1 µM), CNQX (20 µM), and nifedipine (10 µM) (all from Sigma) was added to the cultures that were incubated for 48 h before microscopic analysis. To evaluate the axonal outgrowth of the explants, neurite density was analyzed 100 µm in front of the concave side of the explant with ImageJ. Each data point represents measurements in 1 entorhinal slice culture. Axon outgrowth was normalized to the mean value of the corresponding PRG-1-expressing slices. [...] Experiments were performed according to established protocols (; ). Briefly, after 3 days in vitro, (DIV3) or after DIV7, respectively, cultures were fixed with 4% paraformaldehyde (PFA), resliced, coverslipped, and imaged with a fluorescence microscope (Olympus, BX 50) equipped with a Cool SNAP ES digital camera (Roper Scientific). To measure axonal ingrowth, fluorescence intensity of the GFP-positive EC projection was determined in the ML using the software ImageJ or MetaMorph (Molecular Devices). Two regions of interest (ROIs, all ROIs were identical) comprising the full width of the ML were randomly selected in the ML and in the DG. The average fluorescence in the DG was used to determine the background fluorescence, and the difference in fluorescence intensity between the ML and the DG reflected the specific fiber ingrowth in the ML. Data were normalized to the fluorescence intensity of fiber ingrowth of WT→WT (Fig. f,g). […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Chemicals Glutamic Acid