For immunofluorescence analysis, primary neurons seeded on coated coverslips were processed and confocal imaging was performed using a Nikon A1 confocal laser scanning microscope, as previously described [26 (link)].
Primary antibodies to detect βTubulin III (Millipore, CA, USA), pTauser422 (OriGene Technologies, MD, USA), and 6E10 (Bio Legend, CA, USA) were used following datasheet recommended dilutions. Alexa secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) were used at 1 : 200 dilution.
The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections. The image rendering was performed by Adobe Photoshop software.
For apoptosis detection, after 3 washes with 50 μl of binding buffer (10 mM HEPES, pH 7.5, containing 140 mM NaCl, and 2.5 mM CaCl2), the specimen was incubated for 15 minutes with 50 μl of double staining solution (binding buffer containing 0.25 μl of annexin V-FITC and 0.25 μl of propidium iodide (PI); BD Pharmingen™, Erembodegem, Belgium). Finally, the specimen was washed 5 times with 50 μl of binding buffer, mounted with 15 μl of binding buffer, and visualized under fluorescence microscopy [24 (link)].
Primary antibodies to detect βTubulin III (Millipore, CA, USA), pTauser422 (OriGene Technologies, MD, USA), and 6E10 (Bio Legend, CA, USA) were used following datasheet recommended dilutions. Alexa secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) were used at 1 : 200 dilution.
The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections. The image rendering was performed by Adobe Photoshop software.
For apoptosis detection, after 3 washes with 50 μl of binding buffer (10 mM HEPES, pH 7.5, containing 140 mM NaCl, and 2.5 mM CaCl2), the specimen was incubated for 15 minutes with 50 μl of double staining solution (binding buffer containing 0.25 μl of annexin V-FITC and 0.25 μl of propidium iodide (PI); BD Pharmingen™, Erembodegem, Belgium). Finally, the specimen was washed 5 times with 50 μl of binding buffer, mounted with 15 μl of binding buffer, and visualized under fluorescence microscopy [24 (link)].
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