Mouse strains and cuprizone administration
CD1 mice were purchased directly from Charles River Laboratories (strain code 022) and C57BL/6J mice were purchased from the Jackson Laboratory (stock No. 000664). Both strains of mice were purchased at 8 weeks of age and kept in a pathogen-free facility. Mice were allowed 1 week of acclimation to the environment upon arrival, and then fed with a 0.2% cuprizone-containing diet (Catalog No. TD.01453, Envigo) or a control diet without cuprizone (Catalog No. TD.00217, Envigo). Feeding was ad libitum for a duration ranging from 4 to 7 weeks. For analysis of cuprizone-containing diet intake, both body weights (recorded three times weekly) and food consumption (recorded daily) were closely monitored.
Tissue sample preparation
For histology and immunohistochemistry, mice were deeply anesthetized by ketamine (Henry Schein, Melville, NY) injection, and transcardially perfused with saline and then with 4% paraformaldehyde (PFA) solution. Mouse brains were then collected and placed in 4% PFA solution for overnight fixation, followed by incubation in 30% sucrose containing phosphate buffered saline (PBS) for cryoprotection. Mouse brains were then frozen in O.C.T. (VWR, Radnor, PA) and cut at 14 μm thickness with a cryostat (Leica, Buffalo Grove, IL). Coronal brain sections were collected focusing on the corpus callosum above the fornix, which is approximately between bregma −0.58 and −0.82 mm (The Mouse Brain in Sterotaxic Coordinates [11]) (Fig. 1a). For any of the subsequent analyses, including histology and immunohistochemistry, at least 3 sections were stained per mouse and the average luxol fast blue myelin scoring or cell count/mm2 was taken to represent a single mouse.
For western blot analysis, mice were sacrificed by cervical dislocation and mouse brains were collected and placed in a 1 mm brain matrix (Alto Acrylic). A 2 mm section of the corpus callosum tissue overlying the fornix (approximately between bregma 0.14 and − 1.86 mm) was dissected out and frozen at −80 °C. Lysis of the tissue was carried out by homogenizing in diluted NP40 Cell Lysis buffer (ThermoFisher, Bridgewater, NJ) in the presence of protease inhibitor cocktail (1:200, Sigma, St. Louis, MO) and protein concentration was quantified using a BCA protein assay kit (Pierce, Waltham, MA).
Black gold staining and Luxol fast blue–periodic acid Schiff (LFB–PAS) stain
Black gold stain was carried out using Black-Gold II compound (Histo-Chem, Jefferson, AR) following the manufacturer’s instructions. LFB staining was carried out using LUXOL FAST BLUE—PAS kit (Hitobiotec, Kingsport, TN) following the manufacturer’s instructions followed by PAS counterstaining with a PAS kit (Sigma, St. Louis, MO). Sections were eventually dehydrated with graded ethanol and mounted with permount (Fisher Scientific, Waltham, MA). Images of the midline of the corpus callosum were taken using Virtual Slide microscope 120 (Olympus, Center Valley, PA). For analysis, the sections were scored blind on a scale of 0–3 (Additional file 1: Figure S1) by judging the relative intensity of blue (myelin content) and pink (demyelinated area).
Immunohistochemistry
For visualization of mature oligodendrocytes, sections were boiled in citrate buffer solution (10 mM, pH 6.0) for antigen retrieval, followed by 1 h blocking in PBS solution containing 10% goat serum and 0.3% Triton and incubated overnight with glutathione S-transferase Pi (GST-π) antibody (Enzo Life Sciences, Farmingdale, NY) at 4 °C. Sections were then rinsed in PBS for washing and further incubated for 3 h in fluorescent secondary antibody (Alexa Fluor Goat anti Rabbit 543, ThermoFisher, 1:200) and to-pro-3 (1:1000, Thermo Fisher, T3605) for nuclear stain.
For visualization of OPCs, sections were blocked with 30% goat serum/0.3% triton in PBS for 1 h, followed by overnight incubation with NG2 antibody (1:750, Millipore, Billerica, MA) at 4 °C. After rinsing in PBS, sections were then incubated with Alexa Fluor goat-anti-rabbit secondary antibody (1:200, Thermo Fisher) together with to-pro-3 (1:1000, Thermo Fisher, T3605) for nuclear stain.
For visualization of microglia/macrophages, sections were blocked with 5% goat serum and 0.3% Triton in PBS for 1 h, followed by overnight incubation at 4 °C in rabbit-anti-Iba1 (1:500, Wako, Richmond, VA). After rinsing in PBS, secondary antibody incubation was carried out using Alexa Fluor goat-anti-rabbit 488 (1:200, Thermo Fisher). To-pro-3 was also added for nuclear stain (1:1000, Thermo Fisher).
For visualization of astrocytes, sections were boiled in citrate buffer solution for antigen retrieval, followed by blocking in 0.1% Triton/2% goat serum containing PBS for 1 h. Glial fibrillary acidic protein (GFAP) antibody (1:200, ThermoFisher) was used as primary antibody for overnight incubation at 4 °C. After rinsing (3 × 5 min) in PBS, secondary antibody incubation was carried out using Alexa Fluor goat-anti-rat 488 (1:400, ThermoFisher) together with to-pro-3 for nuclear stain.
Finally, slides mounted with clear-mount (Electron Microscopy Sciences, Hatfield, PA) were examined and fluorescence images of the midline of the corpus callosum were taken using a three-channel confocal microscope system (Eclipse C1, Nikon, Melville, NY). For all cell counting, positively stained cells are identified by antibody-to-pro-3 colocalization (Additional file 1: Figure S2).
Western blot
For analysis of myelin basic protein (MBP), myelin-associated glycoprotein (MAG), ionized calcium-binding adapter molecule 1 (Iba1) and Glial fibrillary acidic protein (GFAP) levels, both CD1 and C57BL/6 tissue lysates (6 μg) were combined with 5× Laemmli loading buffer containing 0.1% bromophenol blue, 7.7% Dithiothreitol (DTT), 10% SDS, 50% Glycerol and 60 mM Tris–Cl (pH 6.8). Samples were denatured by boiling and then loaded (6 μg/lane) and analyzed in a 12% acrylamide protein gel. Upon completion of gel electrophoresis, protein was wet transferred onto 0.45 μm nitrocellulose membrane (Bio-Rad), followed by blocking in 5% BSA solution and incubation at 4 °C overnight with primary antibodies against MBP (mouse-anti-MBP, 1:200, Serotec, Hercules, CA), MAG (rabbit-anti-MAG, 1:1000, Santa Cruz, Dallas, TX), Iba1 (rabbit-anti-Iba1, 1:1000, Wako), GFAP (rabbit-anti-GFAP, 1:10000, Abcam, Cambridge, MA) and beta-tubulin (mouse-anti-beta-tubulin, 1:5000, Sigma) for loading control. After washing, fluorescent secondary antibody (Li-cor goat-anti-mouse IRDye 680 for MBP and beta-tubulin and Li-cor goat-anti-rabbit IRDye 800 for Iba1, GFAP and MAG) incubation was performed for 1 h at room temperature, followed by detection of fluorescence signal using odyssey imaging system (Li-cor, Lincoln, NE).
For analysis of NG2 protein levels, 10 μg of protein samples were loaded and run in a 6% acrylamide protein gel. Primary antibody (Rabbit-anti-NG2, 1:1000, Millipore) and secondary antibody (HRP-linked anti-Rabbit IgG, 1:5000, Cell signaling, Danvers, MA) incubation was performed similarly, and detection of chemiluminescence signal was performed using ECL prime reagent (GE healthcare) and GeneGnomeXRQ imaging system (Syngene, Frederick, MD).
Statistical analysis
Statistical differences between the group data of C57BL/6 and CD1 mice were analyzed using either unpaired student’s t-test or two-way ANOVA analysis where appropriate. Statistical differences between cuprizone treated and control groups within individual strains were analyzed using student’s t-test. Differences were considered to be significant at P < 0.05 and data are presented as the mean ± standard error of the mean (SEM).