Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Abstract

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares. Video LinkThe video component of this article can be found at https://www.jove.com/video/2568/ Protocol 1. Neuron transfection 1. Culture embryonic day 18 (E18) rat hippocampal neurons on poly-d-lysine-coated MatTek 35-mm glass-bottom dishes 1 . On 16-18 days in vitro(DIV), transfect neurons using the Clontech CalPhos Mammalian Transfection Kit. First, replace the culture medium with 1.5 ml Dulbecco's Modified Eagle Medium (DMEM) per 35-mm dish 0.5 hour prior to transfection. Save the original culture medium in a sterile 15 ml tube for later (step 1.6) use. 2. Mix 10 μg pEGFP-N1 plasmid DNA with sterile H 2 O (Clontech) and 12.4 μl 2 M calcium solution (Clontech) to a total volume of 100 μl. 3. Add the mixture from step 1.2) to 100 μl 2×HBS dropwise while vortexing 2×HBS at medium speed. 4. Let the mixture sit at room temperature for 20 minutes and then add the final mixture from step 1.3) into DMEM-incubated neurons. 5. Put the neurons back into the 37 °C incubator for 1-1.5 hours. 6. Remove the calcium phosphate-containing medium, then wash cells with DMEM three times. Before returning the culture dish to the incubator, exchange the DMEM medium with the original culture medium. FRAP experiment on a spine1. Neurons are used for the FRAP experiment two to four days after transfection. 2. Replace the culture medium from the 35-mm glass-bottom dish, by immediately adding pre-warmed Tyrode Solution, which contains (in mM) NaCl 145, KCl 5, HEPES 10, Glucose 10, Glycine 0.005, CaCl 2 2.6, and MgCl 2 1.3 (pH adjusted to 7.4 with NaOH). 3. A Zeiss LSM 710 confocal microscope is used for the FRAP experiment. The Zeiss TempModule system is used to control the temperature (37°C), the humidity and the CO 2 (5%) of the working system. Make sure that the CO 2 tank is connected and the water bottle, which is used for balancing humidity, is filled with water. 4. Find a transfected mature dendrite with the 100×objective (αplan-APOCHROMAT 100×/1.46 oil). If the transfected cells in the dish are sparse, search for a desired cell with the 40× objective (plan-NEOFLUAR ...

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