Computational protocol: Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control

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[…] Spinal cords were obtained from embryonic day 12–14 fetal rats. After removal of the dorsal root ganglia, 10–14 spinal cords were dissected, washed with 5 ml of Earl's Balanced Salt Solution, and centrifuged for 2 min at 150 g. The tissue was resuspended and incubated for 15 min at 37°C with 0.02% trypsin followed by addition of DNase I (80 µg/ml final) and trypsin inhibitor (0.52 mg/ml). Digested tissues were mechanically dissociated and centrifuged at 150 g for 10 min. The dissociated cells were plated at a density of 12 × 104 cells/cm2 in MEM (Invitrogen) supplemented with 10% heat-inactivated FBS or in Neurobasal media (Invitrogen) supplemented with B27. Rat Purkinje cells were obtained from postnatal day 7 rat cerebellum as described previously ().Hippocampal neuron cultures were prepared from an embryonic day 19 mouse. The brains were removed and hippocampi were freed of meninges, treated with 0.025% trypsin, minced, and plated on poly-l-lysine (Sigma-Aldrich) wells in Neurobasal supplemented with 2% B27. Neurons were maintained at 37°C and 5% CO2. For drug treatment, cells were treated at 16–18 d in vitro (DIV) before fixation with 10 µg/ml actinomycin D for 3 h (ActD; Sigma-Aldrich) or with 100 µM DHPG for 5 min (Sigma-Aldrich). Cells were transfected with calcium phosphate precipitations at 10 DIV. In brief, 4 µg DNA were mixed with calcium chloride and precipitated with HBSS phosphate buffer (PB). The microprecipitates were applied to the cells for 45 min, and then washed off. Cells were fixed and stained after 20 h of expression.For immunofluorescence analysis, cells were fixed with 2% PFA, permeabilized with 0.2% Triton X-100, and blocked with 10% FBS in PBS. Fixed cells were incubated for 1 h at room temperature with the primary antibodies diluted in 10% FBS in PBS. The cells were washed three times with PBS and incubated for 1 h at room temperature with Cy2-conjugated α-mouse IgG and Cy3-conjugated α-rabbit IgG secondary antibodies (GE Healthcare) and washed again three times with PBS. Where applicable, cells were stained with 50 µg/ml phalloidin-FITC conjugate (Sigma-Aldrich) in PBS for 25 min at RT and washed three times. Transfections were counterstained using Pacific blue–conjugated secondary antibodies. Coverslips were embedded in Mowiol (EMD) or Gelmount (Biomeda). Images were recorded using the confocal scanning microscope (LSM 510 [Carl Zeiss, Inc.], 63/1.4 objective, or Radiance 2100 [Bio-Rad Laboratories], 60/1.4 objective) and analyzed using the Carl Zeiss, Inc. software as well as the ImageJ program (version 1.40). The mask to identify active synapses and surrounding areas was generated on the basis of spatial correlation of the synaptophysin and the phalloidin staining, large accumulations were discarded from analysis to prevent misinterpretations. In brief, an algorithm was realized with the ImageJ package and the JACoP plug-in (; http://rsb.info.nih.gov/ij/) that takes the overlap of presynaptic and postsynaptic signal and enlarges it three times by one pixel. In this way, areas within ∼400 nm or less were identified as synapses. [...] Three adult male rats weighing 200–250 g were transcardially perfused under deep anesthesia (60 mg/kg Nembutal i.p.) with 150 ml of 0.9% saline at room temperature followed by 200 ml of cold 4% PFA in 0.1 M PB, pH 7.4. Brains were dissected, postfixed for 2 h at room temperature, and cryoprotected in 30% sucrose/PB at 4°C. They were then frozen with dry ice and cut into 40-µm transverse sections with a freezing microtome. All antibody solutions were prepared in PB and 0.3% Triton X-100 and incubated overnight at room temperature. After incubation with the cocktail of primary antibodies, the sections were washed three times in PB and subsequently incubated for 2 h at room temperature in a cocktail of Cy2-conjugated donkey α-mouse IgG and Cy3-conjugated donkey α-rabbit IgG secondary antibodies (1:100 each; Jackson ImmunoResearch Laboratories). Sections were washed three times in PB, mounted on gelatin-coated slides, and coverslipped in Gelmount (Biomeda). Anatomical boundaries and nomenclature were named according to . Images were acquired on a confocal microscope (10/0.3, 20/0.5, and 63/1.4 objectives) and exported in JPEG format; the contrast and brightness were adjusted and final plates were composed with Adobe Illustrator 9 or Corel Draw 9. […]

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