Computational protocol: Arc Interacts with the Integral Endoplasmic Reticulum Protein, Calnexin

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Protocol publication

[…] PLA was performed using the Duolink PLA Kit with red (DUO92008) or orange (DUO92007) detection reagents, anti-mouse minus probe (DUO92004), and anti-rabbit plus probe (DUO92002). Manufacturer’s instructions were followed for cultured neurons except that Roche blocking solution was used. For F-actin staining, phalloidin-FITC (Sigma; 0.5 μg/mL) was added in the penultimate wash step (wash buffer B) for 10 min. On brain sections, antigen retrieval was performed as above, all incubation times and wash steps were doubled, and the blocking buffer consisted of PBS containing 5% horse serum and 5% bovine serum albumin.Images were taken on a Leica SP5 Laser Scanning confocal microscope. Immunofluorescence of cultured neurons was imaged with a 63× objective, a 561 nm laser for Alexa Fluor 568, a 633 laser for Alexa Fluor 647, and a 402 laser for DAPI. Two optical sections were imaged—one at the dendritic level and the other at the equatorial plane of the nucleus. Dendritic PLA and phalloidin-FITC staining were imaged using a 100× objective. For PLAs of cultured neurons, 24 z-stacks of 30 optical sections were taken from each coverslip using a 40× objective, 402 nm excitation for DAPI, and 598 nm excitation for the red PLA signal or 561 nm for the orange PLA. For PLA on brain sections, tile scans were taken at 40× of 5× 4 z-stacks of 14 optical sections, covering the DG.Confocal stacks were analyzed with Imaris (Bitplane, RRID:SCR_007370) using the 3D “spot” function for PLA spots and “surface” function for nuclei. Optimal parameters for these functions were determined using the negative control (no antibodies) and positive control (Arc/Arc) from each experiment and kept constant for all images belonging to this experiment. In cell culture, PLA was quantified as the number of spots divided by the number of cells. In brain sections, the volumes of the molecular and granule cell layers (GCLs) were measured by creating a “surface object”, defined by the lateral boundaries of the layers in XY direction, and the tissue surface in Z direction. PLA was quantified as spot density (PLA/mm3) inside this defined volume. All analyses were performed by investigators blind to the experimental conditions. [...] SH-SY5Y human neuroblastoma cells were plated on 1 cm coverslips coated with collagen at a concentration of 0.3 μg/mL and were maintained in complete DMEM. Cells were transiently transfected at 70% confluence using Lipofectamine 2000 (Invitrogen) and 2 μg of Arc-GFP, calnexin C-terminus-mCherry, or both. Transferrin uptake assays were performed 24 h after transfection. Cells were serum-starved for 6 h prior to experiments (DMEM + 0.5% FBS). Alexa Fluor 647-conjugated Transferrin (Life Technologies T-23366) was added to each sample to a final concentration of 15 μg/mL for 30 min on ice. The samples were then returned to 37°C for 15 min to restore endocytosis. Cells were immediately placed on ice, washed twice with cold PBS, and fixed using 1% PFA on ice for 30 min. Coverslips were mounted using Prolong Gold anti-fade reagent with DAPI.All cells were imaged on a Leica SP2 confocal microscope using a 60× objective. Identical settings were used for each sample. Images were analyzed in ImageJ (RRID:SCR_003070). Endocytosis was quantified by integrating the background-subtracted Alexa Fluor 647 signal within the boundaries of each cell. Transfected cells within each sample, determined by the presence of GFP (Arc), mCherry (calnexin), or both, were compared to internal controls (untransfected cells from the same slide, in the same field of view), and conditions were compared to each other using ANOVA after first normalizing to internal controls for each replicate. All analyses were performed by investigators blind to the experimental conditions. […]

Pipeline specifications

Software tools Imaris, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Chemicals alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid, Glutamic Acid