C8484

Cell were grown on 13 mm Nunc Thermanox coverslips. After treatments, cells were fixed with Karnovsky’s fixative containing 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight at 4ºC. Cells were then washed three times with 0.1 M cacodylate buffer, post-fixed in 2% osmium tetroxide for 1 hour at room temperature, washed, enblock stained in 2% aqueous uranyl acetate at room temperature, washed, dehydrated in a 50–100% ascending series of acetone, infiltrated and embedded in UltraBed resin (Electron Microscopy Sciences). Ultrathin sections (<95 nm silver to gold) were cut using ultramicrotome, examined with a CM 100 TEM (FEI Company) at 100 kV, and images captured with a Hamamatsu C8484 digital camera using a AMT image capture system.

For immunofluorescence microscopy, cells were grown to mid-log phase at the indicated temperatures and fixed using paraformaldehyde and glutaraldehyde as previously described (Levin, 2002 ). Briefly, 500 µL of mid-log phase cells were fixed in paraformaldehyde and glutaraldehyde for 15 minutes at room temperature followed immediately by incubation for 30 minutes on ice. Cells were washed 3 times with 1X PBS and resuspended in 1X GTE. FtsA was stained using 1:500 anti-FtsA primary antibody and 1:200 secondary anti-rabbit IgG conjugated to Alexa Fluor 488 (Life Technologies) or DyLight 550 (Pierce). Anti-FtsZ was stained with 1:2000 anti-FtsZ primary antibody and 1:200 of the Alexa Fluor 488 or DyLight 550 conjugated goat-anti-rabbit IgG secondary antibody. Fluorescence images were captured using an Olympus BX60 microscope with a 100X oil immersion objective and a Hamamatsu C8484 digital camera with HCImage software. Images were compiled using Adobe Photoshop.

Animals were sacrificed at P35 or P105 after either receiving CNO for 2 weeks or not. hM4D (mCherry-tagged) or GFP-infected thalamic cells were identified by their fluorescence at 40x magnification under infrared and diffusion interference contrast microscopy using an inverted Olympus BX51W1 microscope coupled to a Hamamatsu C8484 camera. Intrinsic and active membrane properties (resting membrane potential, input-output firing frequency curve) were recorded in current clamp using the K-Gluconate intracellular solution detailed above before and after 10μM CNO was bath applied to the slice.

L5 DRGs were dissected from adult rats and then fixed using 4% paraformaldehyde for 4 hrs at room temperature (RT). DRGs were next transferred into a 30% sucrose solution and left at 4°C until sinking of the tissues could be observed (~3 days). Tissues were cut at 10 μm thickness using the Bright OTF 5000 Microtome Cryostat (Hacker Instruments and Industries, Inc., Winnsboro, SC), and fixed onto gelatin coated glass slides and kept at −20°C until use. DRG slices were permeabilized and saturated using PBS containing 3% BSA, 0.1% triton X-100 solution for 30 min at RT, and then anti-neurofibromin C-terminal (Abcam Cat# ab17963) was added overnight. The slices were then washed 3X in PBS, and incubated with PBS containing 3% BSA, 0.1% triton X-100 containing secondary antibody (Alexa 594 goat anti-rabbit (Life Technologies)) for at least 3 hrs at RT. After 3 washes (PBS, 10 min, RT), either DAPI was used to stain the nuclei of cells. Slides were mounted and stored at 4°C until analysis. Immunofluorescent micrographs were acquired on an Olympus BX51 microscope with a Hamamatsu C8484 digital camera using a 20X UplanS Apo 0.75 numerical aperture objective. For a given experiment, all images were taken using identical acquisition parameters by individuals blinded to the treatment groups.