Alonso M. Higuero, Lucía Sánchez-Ruiloba1, Laura E. Doglio, Francisco Portillo1, José Abad- Rodríguez, Carlos G. Dotti and Teresa Iglesias. Kidins220/ARMS modulates the activity of microtubule-regulating proteins and controls neuronal polarity and development. JBC Papers in Press. Published on November 10, 2009 as Manuscript M109.024703.
...Tuj 1 (Neuron-specific class III beta-tubulin)-Mouse (MO15013, Neuromics Antibodies, Edina, MN)...
Immunofluorescence Method:
Cells grown on coverslips were fixed for 5 min in 4% paraformaldehydecontaining 4% sucrose in phosphate buffer saline (PBS) at 37ºC. Cells were then permeabilized with 0.2% Triton X-100 in PBS during 5 min at room temperature. After blocking (5% bovine serum albumin in PBS for 1 h), cells were incubated with the corresponding primary antibodies, and immunoreactivity was
detected with the suitable fluorophore-conjugated secondary antibody before mounting on slides with Mowiol4-88 (Harland Co., UK). Confocal images were acquired using an inverted Leica TCS SP5 laser confocal microscope with a 63X Plan- Achromatic oil immersion objective and processed with LAS AF Leica Application Suite and Adobe Photoshop CS2 (Adobe Systems
Inc., CA). All images correspond to the projection of sections from a ~50μm z-stack, except for colocalization analysis where they correspond to 0.5-0.7μm single sections.
Cells grown on coverslips were fixed for 5 min in 4% paraformaldehydecontaining 4% sucrose in phosphate buffer saline (PBS) at 37ºC. Cells were then permeabilized with 0.2% Triton X-100 in PBS during 5 min at room temperature. After blocking (5% bovine serum albumin in PBS for 1 h), cells were incubated with the corresponding primary antibodies, and immunoreactivity was
detected with the suitable fluorophore-conjugated secondary antibody before mounting on slides with Mowiol4-88 (Harland Co., UK). Confocal images were acquired using an inverted Leica TCS SP5 laser confocal microscope with a 63X Plan- Achromatic oil immersion objective and processed with LAS AF Leica Application Suite and Adobe Photoshop CS2 (Adobe SystemsInc., CA). All images correspond to the projection of sections from a ~50μm z-stack, except for colocalization analysis where they correspond to 0.5-0.7μm single sections.
Image: Rat hippocampal neurons were fixed at 1.5 DIV and immunostained for the neuronal marker βIII-Tubulin/Tuj1 (blue).
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