Reagents and Antibodies
The nuclear dye (Hoechst 33342) and the lipophilic dyes (Fluoromyelin-green and Fluoromyelin-red) were purchased from Invitrogen (Carlsbad, CA, USA). The following antibodies were used in our study, rabbit polyclonal anti-Kv1.2, anti-Kvβ2, anti-Kv1.4, and anti-Kv2.1 antibodies (Alomone Labs, Jerusalem, Israel), anti-Kv1.2 and anti-contactin associated protein 2 (Caspr2) antibodies (Millipore, Billerica, MA, USA), and anti-neurofilament 200 antibody (Sigma, , St Louis, MO, USA), mouse monoclonal anti-Kv1.2, anti-Kvβ2, and anti-Kv1.4 antibodies (clone #: K14/16, K17/70, and K13/31, respectively; UC Davis/NIH Neuromab Facility, Davis, CA, USA), anti-β-tubulin (Millipore), rat monoclonal anti-myelin basic protein (MBP) antibody (Chemicon, Temecula, CA, USA), goat polyclonal anti-GFAP and anti-NG2 antibodies (AbCAM, Cambridge, MA, USA), and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MEVGWYRSPFSRVVHLYRNGK) was purchased from Pro-Spec (Rehovot, Israel), and proteolipoprotein (PLP) peptide 139-151 (HCLGKWLGHPDKF) from Anaspec (Fremont, CA, USA).
Induction of chronic EAE
Complete Freund's Adjuvant (CFA) was prepared by adding ground inactivated mycobacteria tuberculosis H37Ra (Difco Laboratories, Detroit, MI, USA) to Incomplete Freund's Adjuvant (Difco Laboratories) at 4 mg/mL. CFA was passed through a glass syringe with an equal volume of sterile-filtered PBS (control) or PBS containing myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (1 mg/mL final concentration). 12-week-old female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were immunized with 100 μL of MOG/CFA or CFA only (control) by subcutaneous injection at four sites in the belly and in each hind footpad. Pertussis toxin was injected at 0 and 2 days post immunization (DPI). Thy1:YFP transgenic mice (Jackson Laboratories) were maintained on a C57BL/6 background, and thus were immunized with the same protocol. Control mice received the same injection of CFA/PBS without the peptide. The chEAE experiment was performed 5 times, 10-17 mice per experiment. Clinical scores were assigned on a scale of 0-6 [0 = no symptoms, 1 = loss of tail tone, 2 = hindlimb paresis, 3 = moderate paralysis, 4 = paraplegia (complete hindlimb paralysis), 5 = quadriplegia, 6 = death or moribund state]. Grade 6 animals were removed from the study.
Induction of remitting-relapsing EAE
Solution preparation and immunization of 12-week-old female SJL/J mice (Jackson Laboratories) for rrEAE were performed identically to the chEAE procedures except for the use of PLP peptide 139-151. Control mice received the same injection of CFA/PBS without the peptide. The rrEAE experiment was performed 3 times, 12-15 mice per experiment.
Cardiac perfusion and tissue fixation
Animals were deeply anesthetized with 250 mg/kg avertin (12.5 mg/mL 2,2,2-tribromoethanol dissolved in ddH20/0.025% 2-methyl-2butanol) (Sigma). The thoracic cavity was opened to expose the heart, and the mice were perfused with 20-30 mL ice-cold PBS followed by 20 mL 4% formaldehyde in PBS. The brain and spinal cord were carefully removed and post-fixed overnight in 4% formaldehyde in PBS. The brain was cut into 3-mm blocks using an acrylic brain matrix (Braintree Scientific, Braintree, MA, USA) and placed in 30% sucrose for at least 24 hr before sectioning.
Sectioning of brain and spinal cord
Brain and spinal cord tissues were arranged in the same block, embedded in optimal cutting temperature (OCT) media (Sakura Finetek USA, Inc., Torrance, CA, USA), and stored at -80 °C until sectioning. The spinal cord was cut into segments and arranged for both transverse and longitudinal sectioning. The tissue blocks were cut with a Microm HM550 cryostat (Thermo Scientific, Waltham, MA, USA) and the 40-μm sections were collected on Superfrost Plus microscope slides (FisherScientific, Pittsburgh, PA, USA) for storage at -20°C.
Sections were incubated in PBS/0.2% Triton X-100 for 1 hr at room temperature to permeabilize the tissue, then blocked with 2.5% normal goat or donkey serum (matched with the host species of the secondary antibody) for 1 hr at room temperature. The primary antibodies were then added in blocking solution, and the sections were incubated for 3 hr at room temperature, then overnight at 4°C. The next day, the sections were rinsed 10 × 5 min at RT, the appropriate secondary antibody was added in blocking solution, and the sections were incubated for 3 hr at RT. Then, the sections were incubated in Hoechst 33342 and/or Fluoromyelin for 10 min at RT. Sections were rinsed 10 × 5 min at RT and coverslipped using tris-buffered Fluoro-Gel mounting media (Electron Microscopy Sciences, Hatfield, PA, USA).
Fluorescence light microscope and image capture
Fluorescence images were captured with a Spot CCD camera RT slider (Diagnostic Instruments, Sterling Heights, MI, USA) in a Zeiss upright microscope, Axiophot, using Plan Apo objectives, 2.5×/0.075, 10×/0.30, and 20×/0.50, saved as 16-bit TIFF files, and analyzed with MetaMorph (Molecular Devices, Downingtown, PA, USA) and Sigmaplot 11.0 (Systat Software, Inc., Chicago, IL, USA) for fluorescence intensity quantification. Exposure times were controlled so that the pixel intensities in the tissues of interest were below saturation, and the same exposure time was used within each group of an experiment. Image brightness and contrast were adjusted using Adobe Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA, USA). All fluorescence intensity measurements were taken from the original captured images.
Areas of interest were outlined with the region measurement tool in MetaMorph by tracing the borders of the lesions as shown by Hoechst staining. The traced region was then overlaid onto the Kv channel image, and the average intensity of the region was recorded. For control images, the average intensity of the spinal cord white matter was measured. The background intensity of the slide was measured and subtracted from region measurements for each image. For measurement of cell processes, the line measurement tool was used to trace each Kv channel-stained process directly, and the average fluorescence intensity along this line was recorded. Measurements of non-lesioned white matter regions of the same image were obtained, and were subtracted from the measurements of cell processes. For motor neurons, the somatodendritic region revealed by Kv2.1 staining was outlined and the average fluorescence intensity was recorded. Gray matter background intensity was measured for each image and subtracted from the intensity of the cells. For each experimental group, the same immunostaining and imaging procedures were used so that the fluorescence intensities can be compared within the group.
High-magnification confocal images were captured with a Leica TCS SL confocal imaging system (Leica Microsystems, Mannheim, Germany), using a 100× HCX Plan Apo CS oil immersion objective (numerical aperture = 1.40). Multiple channels were acquired simultaneously, and the signal was averaged over six scans. Channel crosstalk was largely eliminated through optimization of the laser line intensity by acousto-optical tunable excitation filters, and by spectral detectors allowing precisely-defined bandwidth adjustment. Images were saved as 8-bit TIFF files and adjusted for brightness and contrast using Adobe Photoshop 7.0.
Mice were killed with carbon dioxide, and brain and spinal cord tissues were quickly removed, flash-frozen in liquid nitrogen, and stored at -80°C until use. The tissue samples were then weighed and homogenized in 1:4 (w/v) Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA, USA) with 5% β-mercaptoethanol (Sigma). Protein samples were resolved by SDS-PAGE and transferred to a PVDF membrane (GE Healthcare, Piscataway, N, USA) for western blotting. The membrane was rinsed in TBST (50 mM Tris-Cl (pH 7.5), 150 mM NaCl, and 0.1% Tween20), incubated in 5% dry milk/TBST blocking solution for 1 hr at RT, and then incubated with the primary antibody in blocking solution overnight at 4°C. Membranes were then rinsed in TBST 4 × 10 min, incubated with a horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 hr, and rinsed in TBST 5 × 10 min. Membranes were then incubated in ECL Plus (GE Healthcare) chemiluminescent solution for 5 min, wrapped in plastic, and exposed to X-ray film (Kodak, Rochester, NY, USA). Developed film was digitized with an Epson 3590 scanner (Epson America, Inc., Long Beach, CA, USA). The total intensities of protein bands were measured and quantified with NIH ImageJ. Background was subtracted for each band. The β-tubulin loading ratios were obtained by normalizing against the control band. The Kv channel alterations were obtained by first normalizing against their controls and further normalizing with the β-tubulin loading ratio. All Western blotting experiments were performed three times.
Hippocampal neuron culture and transfection
The hippocampal neuron culture was prepared as previously described from rat hippocampi at the embryonic day 18 (E18) (Xu et al., 2007
; Barry et al., 2010
; Xu et al., 2010
). The neurons were transfected with cDNA constructs at 5 DIV (day in vitro
), fixed at 7 DIV, and stained with a rabbit polyclonal anti-Kv1.4 antibody. YFP-Kv1.2 was previously described (Gu and Gu, 2011
; Gu et al., 2012
). YFP-Kv1.4 was made by inserting the coding sequence of yellow fluorescence protein into the N-terminal region of rat Kv1.4 between G58 and G59 with an engineered NheI site. The construct was confirmed with sequencing.
For both immunofluorescence imaging and Western blotting data, we performed One-Way ANOVA when comparing 2 or 3 experimental groups to one control group, and unpaired t-test when comparing two groups, using Sigmaplot 11.0. Results are provided as mean ± SEM.