Computational protocol: Geniposide Alleviates Amyloid-Induced Synaptic Injury by Protecting Axonal Mitochondrial Trafficking

Similar protocols

Protocol publication

[…] The brain tissue or neuron samples were lysed in 10 volumes (w/v) of radio-immunoprecipitation assay buffer containing a cocktail of complete protease and phosphatase inhibitors and were centrifuged at 15,000 × g for 10 min at 4°C. The protein concentration of the supernatant was determined using the BCA method. Proteins were examined by Western blot analysis using standard protocols. Equal amounts of proteins were loaded and resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were incubated in blocking solution (5% skim milk in PBST, 20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) at room temperature for 1.5 h. The membranes were incubated and gently shaken overnight (at 4°C) in PBST containing 5% skim milk and the indicated primary antibodies from among the following: monoclonal rabbit antibodies against c-AMP response element binding protein (CREB, 1:400, Cell Signaling, Beverley, MA, USA), and GAPDH (1:3000, Cell Signaling); polyclonal rabbit antibodies against phosphorylated Ca2+/calmodulin dependent protein kinase II α (p-CaMKIIα, 1:8000, Santa Cruz, CA, USA); and monoclonal mouse antibodies against synaptophysin (1:500, Abcam, Cambridge, UK), CaMKIIα (1:250, Santa Cruz), phosphorylated CREB (p-CREB, 1:400, Cell Signaling), postsynaptic density protein-95 (PSD-95, 1:500, Abcam), and β-actin (1:2000, Santa Cruz). After four washes with PBST, the membranes were incubated for 1.5 h at room temperature with the corresponding secondary antibodies. The membranes were washed four times with PBST and detected with an infrared imaging system (Odyssey). The intensity of the blots was analyzed and compared using NIH ImageJ program. [...] Neurons cultured in glass-bottom dishes with four chambers (CELLview, Greiner, Germany) were fixed in 4% paraformaldehyde for 20 min and washed with PBS after the neurons were incubated with 2 μM preheated CellTracker CM-DiI (Life Technologies, Waltham, MA, USA) for 20 min at 37°C. LAS X software was used to control the laser-scanning confocal microscope (TCS-SPE, Leica, Germany) equipped with a 63× objective and excitation at 543 nm. Images were taken under the confocal microscope, and Z-stacks were gathered at increments of 0.67 μm. To increase the accuracy of the identification of the surfaces of the dendrite shaft and spines, sequence images were deconvolved to reduce point-spread functions by using the adaptive blind 3D deconvolution method (AutoQuant X, Media Cybermedics, Inc., Bethesda, MD) prior to analysis. The output images were obtained with Build Neurites and Build Spines in NeuronStudio software (CNIC, Mt. Sinai School of Medicine, New York, USA) to mark and record all the spines on the selected dendrites. Dendritic protrusions with a clearly identifiable neck attached to the branch of the dendrite composed a spine. The output data included length, neck diameter, and head diameter of each spine, based on which the types of spines were recognized. Spines with a spine length-to-neck diameter ratio <2.0 were defined as of the stubby type. Spines were categorized as mushroom type if they presented a length-to-neck diameter ratio of more than 2.0 and a head-to-neck diameter ratio of more than 1.3. Spines were categorized as thin type if the spines exhibited a length-to-neck diameter ratio of more than 2.0 and a head-to-neck diameter ratio of <1.3 (Du et al., ). Spine density and morphology were measured separately for portions of three to five selected secondary dendrites per neuron. […]

Pipeline specifications

Software tools ImageJ, LAS X, AutoQuant
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Alzheimer Disease, Liver Diseases, Mitochondrial Diseases
Chemicals Adenosine Triphosphate, Calcium