Ab6319

Immunohistochemistry was performed as previously described [53 (link)]. Half of the brain tissue of all of the mice was obtained via perfusion with paraformaldehyde, and the brain tissue was fixed, dehydrated, and embedded in paraffin. The sections were incubated with mouse anti-CNpase (ab6319, Abcam, Cambridge, UK), rabbit anti-PDGFRα (bs-0231R, Bioss, Beijing, China) and the MYRF (A16355, ABclonal, Wuhan, China), at 4 °C overnight. After the antibodies were washed with PBS, sections were incubated with biotin-labeled goat antimouse antibodies (1:200; Boster, Wuhan, China) at 37 °C for 60 min. DAB was used to mark positive signals. The intensity of each section was quantified using ImageJ software.

Cultured cells were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min following a pretreatment with 2% PFA in the culture medium for 30 min at room temperature. After being washed, cells were incubated in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) and 10% FBS with primary antibodies at 4°C overnight. Primary antibodies specific to the following proteins were used: CNPase (mouse IgG₁ 1:250; ab6319; Abcam), GFAP (rabbit IgG 1:1000; ab7260; Abcam), human CD63 (mouse IgG₁ 1:200; 556019; Becton Dickinson), MAP2 (mouse IgG₁ 1:200; M1406; Sigma-Aldrich), nestin (mouse IgG_2a, 1:200; MAB2736; R&D Systems, MN, USA), rat CD63 (mouse IgG₁ 1:200; MCA4754GA; AbD Serotec) and SOX2 (rabbit IgG 1:200; ab97959; Abcam). After cells were stained with Alexa Fluor 546 mouse IgG₁ (1:2000; Life Technologies), Alexa Fluor 546 mouse IgG_2a (1:2000; Life Technologies), Alexa Fluor 546 rabbit IgG (1:2000; Life Technologies), Alexa Fluor 488 mouse IgG₁ (1:200; Life Technologies) and Alexa Fluor 488 rabbit IgG (1:200; Life Technologies), they were observed through a BIOREVO BZ-9000 (Keyence, Osaka, Japan) fluorescence microscope. Hoechst 33342 was used to stain cell nuclei.

The composition of the protein lysis buffer was as follows: 1% SDS, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride and 2 mM Na₃VO₄. Samples of tissues and cells were sonicated on ice, and then lysates were cleared by centrifugation. The lysates were dissolved in SDS sample buffer without 2-mercaptoethanol (Wako) after determination of the protein concentration using a Pierce BCA Protein Assay Kit (Life Technologies).
After being blocked with 5% skimmed milk, membranes were probed with specific primary antibodies to CNPase (1:1000; ab6319; Abcam, Cambridge, UK), copGFP (1:10,000; AB501; Evrogen, Moscow, Russia), flotillin-1 (1:500; 610820; Becton Dickinson), GFAP (1:1000; AB5804; Millipore), GluR1 (1:1000; AB1504; Millipore), human CD63 (1:250; 556019; Becton Dickinson), rat CD63 (1:250; MCA4754GA; AbD Serotec, San Jose, CA, USA), SOX2 (1:1000; ab97959; Abcam), syntaxin 1 (1:1000; S0664; Sigma-Aldrich) and TUJ1 (1:1000; MMS-435P; Berkeley Antibody Company, Berkeley, CA, USA) followed by incubation with peroxidase-conjugated anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA) or rabbit IgG (1:1000; Rockland, Limerick, PA, USA) secondary antibody. β-actin (1:5000; A5441; Sigma-Aldrich) was used as a loading control. Signals were detected using chemiluminescent reagents (ImmunoStar; Wako).

The hippocampus and cerebral cortex tissues were homogenized and centrifuged, and supernatants were extracted. All of the samples were quantitatively analyzed using BCA for total protein content. The 25 ug of protein was run on a 10% gradient SDS–PAGE gel and electrophoretically transferred to polyvinyl difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Nonspecific binding proteins were blocked in 5% milk for 1.5 h at room temperature. Incubated protein bands with primary antibodies were left overnight at 4 °C before the membranes were incubated with antimouse IgG or antirabbit IgG antibodies for 2 h at room temperature; the antibodies were horseradish-peroxidase-labeled. Blots were visualized using an ECL western blotting kit (Advansta). Protein bands were scanned and semiquantified with densitometry using ImageJ software. The primary antibodies used were MBP (10458-1-AP, Proteintech, Wuhan, China), CNpase (ab6319, Abcam, Cambridge, UK), MAG (DF7669, Affinity, Liyang, China), and MOG (12690-1-AP, Proteintech, Wuhan, China).

The retinal sections with the optic nerve were washed with PBS and blocked with blocking buffer (1% bovine serum albumin (BSA), 1% normal goat serum, and 1% Triton X-100 in 1XPBS). The sections were incubated with primary antibodies against Iba1 (1:200, ab178846, Abcam, Cambridge, UK), IL-6 (1:200, ab9324, Abcam, Cambridge, UK), ED-1 (1:50, MCA341GA, Bio-Rad, Hercules, CA, USA), CNPase (1:200, ab6319, Abcam, Cambridge, UK) and Ym1 + 2 (1:50, ab192029, Abcam, Cambridge, UK). After labeling, the sections were immersed in PBS. Next, the sections were incubated with secondary-antibody-conjugated Alexa Fluor dyes (Invitrogen, Waltham, MA, USA) for 1 h. Counterstaining was performed using DAPI (1:500, Sigma, St. Louis, MO, USA). Sections were visualized and photographed with a Zeiss confocal microscope (LSM900, Zeiss, Oberkochen, Germany). For ED-1 and Ym1 + 2, the positive cells were quantified from six images at the lesion site of the optic nerve using ImageJ. For quantitative analysis of CNPase, the CNPase-stained area per DAPI molecule was estimated by ImageJ.

Proteins were extracted from primary neurons and Ols, ipsilateral cortex and corpus callosum. The antibodies used were: rabbit anti‐ferritin (1:1000, Abcam, ab75973), mouse anti‐CNPase (1:1000, Abcam, ab6319), rabbit anti‐GPX4 (1:1000, Abcam, ab125066), mouse anti‐Rasd1/2 (1:500, Santa Cruz, sc‐398,988), mouse anti‐ARA70 (1:500, Santa Cruz, sc‐373,739), rabbit anti‐UCP2 (1:1000; Cell Signaling Technology, 89,326), mouse anti‐β‐actin (1:1000, Proteintech, 66,009‐1‐lg), anti‐rabbit secondary antibody (1:5000, Cell Signaling Technology, 7074), anti‐mouse secondary antibody (1:5000, Cell Signaling Technology, 7076).

For Western blot analysis, cells were lysed in lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, and protease inhibitors, pH 7.5) at 4°C for 30 min. Equal amounts of cell lysate (25 μg) were separated on 4–12% or 10% acrylamide gels (Thermo Fisher Scientific) and transferred onto 0.22 μm PVDF membranes (Millipore). After blocking with blocking buffer (TBST supplemented with 5% nonfat dry milk), membranes were probed with specific antibodies: mouse anti-CNP (ab6319, 1:2,000; Abcam), goat anti-HIV1 p24 (ab53841, 1:2,000; Abcam), rabbit anti-Tom20 (ab186735, 1:1,000; Abcam), rabbit anti-GAPDH (5174S, 1:1,000; Cell Signaling Technology), rabbit anti-Flag (14793S, 1:1,000; Cell Signaling Technology), and rabbit anti-β-tubulin (ab6046, 1:1,000; Abcam). Subsequently, blots were probed with species-specific IRdye secondary antibodies, and visualization was performed using an Odyssey infrared imaging system (Li-COR).