Immunocytochemistry and confocal microscopy
Immunocytochemistry was performed on DRG neurons incubated with vehicle
(0.1 % DMSO) or CBD3063 (20 µM) overnight. Cultured DRG neurons were
fixed using ice-cold methanol for 5 min and then allowed to dry at room
temperature. Fixed cells were rehydrated in PSB and then blocked with
PBS containing 3% bovine serum albumin for 30 min at room temperature.
Cell staining was performed with anti-Cav2.2 (Origene,
Cat# TA308673, Rockville, MD) in PBS with 3% BSA overnight at 4°C. The
cells were then washed thrice in PBS and incubated with PBS containing
3% BSA and secondary antibodies (Alexa 488 Chicken anti-Rabbit (Life
Technologies, Carlsbad, CA)) for 1 h at room temperature. Coverslips
were mounted and stored at 4°C until analysis. Immunofluorescent
micrographs were acquired on a Leica SP8 inverted upright microscope
using a 63X, oil immersion objective. For all quantitative comparisons
among cells under differing experimental conditions, camera gain and
other relevant settings were kept constant. The freeware image analysis
program Image J (http://rsb.info.nih.gov/ij/) was used for quantifying
cellular fluorescence. Regions of interest (i.e. cells) were defined by
hand using Image J.