Mouse anti β tubulin 3

Primary sensory neurons were isolated from 3 to 4-weeks-old Twitcher mice and WT littermates as described (Miranda et al, 2011 (link)). Dissociated neurons were seeded on poly-L-lysine (PLL)/laminin-coated coverslides in 24-well plates at 7,500 cells/well and maintained in culture for 12 h or up to 7 days in DMEM:F12 supplemented with 50 ng/mL nerve growth factor (NGF), 1% Pen/Strep, 1x B-27 and 2mM of L-glutamine. The DRG culture method described produces a mixture of approximately 20% neurons and 80% glial cells (satellite and Schwann cells). As such, in experiments using DRG cultures, neurons were either identified by β-tubulin III staining, as detailed below, or by the distinctive round morphology of cell bodies, that possess protruding neurites, which contrasts with the spindle-shaped morphology of glial cells and fibroblasts. To analyze neurite outgrowth, fixed DRG neurons were permeabilized in 0.2% Triton X-100 and neurites were visualized using mouse anti-β-tubulin III (1:2,000; Promega) and anti-mouse Alexa Fluor 488 (1:1,000; Molecular Probes). All neurites with a length longer than the cell body diameter were measured using ImageJ/NeuronJ in over 100 neurons/condition. The percentage of neurites with axonal swellings was calculated in WT and Twitcher DRG neuron cultures.

After 4 DIV in DMEM:F12 supplemented with NGF, Pen/Strep, B27 and L-glutamine, DRG neurons from Twitcher and WT littermates were grown for 16 h in the same media without NGF after which cells were incubated at 37°C for 20 min in DMEM:F12 with or without 100 ng/mL NGF. After NGF stimulation, cells were fixed in 2% PFA for 20 min at room temperature, followed by immunolabeling with rabbit anti-pTrkA (1:10, Abcam) and mouse anti-β-tubulin III (1:2,000, Promega). Images acquired with a Zeiss Axio Imager Z1 microscope were analyzed using the Fiji software. The ratio of pTrkA positive particles and the cell body area was calculated in at least 20 cells per condition.