Immunostaining of trigeminal and dorsal root ganglia neurons

Trigeminal ganglia from 8–10 week old male and female C57Bl/6 mice were collected and treated with Sema3A at 50 ng/mL. After 3 days in culture, neurons were fixed with 4% PFA for 20 min and carefully washed 3 times with PBS for 5 min each on a slow speed orbital shaker. The dishes containing neurons were halfway filled with PBS and stored at 4 °C until processing for immunofluorescence staining. Dorsal root ganglia from E15 embryos were isolated and seeded on 12 well glass bottom plates. After 2 h incubation at 37 °C, neurons were treated with 50 ng/mL NGF. After 2 days in culture, neurons were fixed with PFA as for TG neurons described above. For immunofluorescence staining, neurons were incubated with blocking buffer (2% BSA, 2.5% donkey serum in PBS) for 1 h at room temperature and then incubated overnight with primary antibodies (see Table ​Table1)1) at 4 °C in same blocking buffer. Neurons were washed 3 times in PBS for 5 min each and incubated at room temperature for 1 h with secondary antibody (see Table ​Table1)1) in blocking buffer. Neurons were washed 3 times with PBS, kept in PBS and imaged at 20 × magnification using an AxioObserver fluorescent microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) and processed with Adobe Photoshop using the same adjustment for all neurons. Neurons treated with secondary antibody alone were tested for background interference and this is shown on Fig. ^S4.

Antibodies used for immunofluorescence staining of trigeminal and dorsal root ganglia neurons.