To express genes of interest, 10–20 µl of purified viral construct was intranasally administered to mice ranging between 10 and 40 days of age. Typically, viral delivery was repeated in three consecutive days. At 10 days post infection, mice were anesthetized with CO2, rapidly decapitated, and entire turbinates and septum were dissected and kept on ice in a Petri dish filled with freshly oxygenated carbogen-modified artificial cerebrospinal fluid (ACSF) that contained (in mM): 120 NaCl, 25 NaHCO3, 3 KCl, 1.25 Na2HPO4, 1 MgSO4, 1.8 CaCl2, 15 glucose, 305 mOsm (adjusted with sucrose), pH 7.4. For imaging, a small piece of the OE was mounted in the perfusion chamber (RC-23, Warner Instruments) with the apical surface facing down and analyzed on the Nikon TiE-PFS-A1R confocal microscope equipped with a 60× oil-immersion objective, using preset configuration for acquisition of GFP and mCherry fluorescence. Image acquisition settings were set to avoid pixel saturation and maintained equal when comparing WT control and INPP5E-KO tissue.
For total internal reflection fluorescence microscopy (TIRF) en face imaging, virally transduced mice were prepared as above. TIRF imaging was performed on a Nikon Eclipse Ti-E/B inverted microscope equipped with a 100× oil immersion CFI APO TIRF 1.49 NA and an EMCCD camera (iXon X3 DU897, Andor Technology).
For total internal reflection fluorescence microscopy (TIRF) en face imaging, virally transduced mice were prepared as above. TIRF imaging was performed on a Nikon Eclipse Ti-E/B inverted microscope equipped with a 100× oil immersion CFI APO TIRF 1.49 NA and an EMCCD camera (iXon X3 DU897, Andor Technology).