After the mice were sacrificed at 20 or 40 dpi, paraffin-embedded tissue sections were cut into 5 μm pieces. The slices used for immunofluorescence staining were incubated in medium containing 10% normal serum. The slices were incubated with an MBP antibody (Abcam, Cambridge, MA, USA, 1 : 400) at 4°C for 2 days. After incubation with a goat anti-rabbit antibody at 37°C for 2 h, the slices were counterstained with 4,6-diamino-2-phenyl indole (DAPI, Southern Biotech, AL, USA). A representative image was obtained using the NIS-Elements BR 3.0 system (Nikon, Tokyo, Japan), and an integrated optical density (IOD) value was calculated for further data analysis.
Mbp antibody
Manufactured by Abcam
Sourced in United States
The MBP antibody is a protein-specific antibody that recognizes the Myelin Basic Protein (MBP). MBP is a structural protein found in the myelin sheath of the central nervous system. The MBP antibody can be used for the detection and analysis of MBP in various research applications.
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Lab products found in correlation
4 6 diamino 2 phenyl indole dapi, by Southern Biotech (1 mentions)
Nis elements br 3.0 system, by Nikon (1 mentions)
Diaminobenzidine dab kit, by Merck Group (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Sucrose, by Merck Group (1 mentions)
Chloral hydrate, by Merck Group (1 mentions)
5 protocols using mbp antibody
1
Quantifying Myelination via MBP Immunofluorescence
2
Western Blot Analysis of Corpus Callosum Proteins
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Protein extract from corpus callosum were centrifuged at 560 g for 5 min; then stored at -20°C. To ensure loading of equal amounts during Western blotting, protein concentration of each sample was determined with Bradford assay. Equivalent amounts of total protein extract from each sample were mixed with sample buffer, boiled, and loaded onto SDS polyacrylamide gels. Electrophoretic separation of the extracts was typically performed on 7.5–15% gel. The proteins were then transferred to polyvinylidene (PVDF) (Gibco, CA) and probed with MBP antibody (1:3000 in blocking buffer) (Abcam, USA) and PLP antibody (1:1000 in blocking buffer) (Abcam, USA) for an overnight on shaker in 4°C. Membranes were subsequently rinsed in blocking buffer (4 times/3 min each time) and stained with secondary antibody (1:1000 in blocking buffer) (Abcam, USA) for 1 hour. Then, membranes were washed with TBS Tween-20 blocking buffer (3 times) and the proteins were visualized with diaminobenzidine (DAB) kit (Sigma, USA). The membranes were scanned and the photos were analyzed with image 2 software.
3
Myelin Debris Phagocytosis by BMDMs
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Myelin debris was isolated from the brains of 2-month-old mice by sucrose density gradient centrifugation as described previously (Grajchen et al., 2020 (link)). Myelin debris was added to the cultured BMDMs at a final concentration of 1 mg/ml. Non-ingested myelin debris was washed away from the cell surface. After removal of non-ingested myelin debris, cells were fixed with 4% PFA, followed by anti-mouse myelin basic protein (MBP) antibody (1/400, Abcam) immunostaining for myelin debris engulfment detection. Images were captured with a fluorescence microscope (Carl Zeiss Axio Observer Z1, Oberkochen, Germany). Oil Red O (ORO) staining was applied to visualize macrophages phagocytosing myelin debris in vitro. Stained samples were captured with a Leica light microscope (n = at least three independent biological replicates).
4
Tracking Transplanted Oligodendrocyte Fate
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At 1 wk after TBI, the rats were anesthetized by i.p. injection of 3.6% chloral hydrate (1 mL/100 g; Sigma-Aldrich). Then, samples were harvested and fixed by paraformaldehyde (PFA) for immunohistochemistry. Samples were put into 4% paraformaldehyde (PFA) phosphate buffer and postfixed until they were processed for immunohistochemistry. The postfixed samples were dehydrated with 20% sucrose (Sigma-Aldrich) at 4 °C overnight, followed by sectioning into 15-µm sections. Then, a 2-step immufluorescent staining method was used to detect the survival and differentiation of the transplanted A2B5+cells emitting GFP (1:1,000; Abcam) fluorescence using an oligodendrocytes 2 (Olig2) antibody (1:500; Abcam). Meanwhile, the expression of MBP was detected using an MBP antibody (1:500; Abcam); simultaneously, we detected the GFP fluorescence to monitor myelinogenesis. The sections were placed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) to observe the implanted cells.
5
Quantifying Axon Regeneration in Nerve Grafts
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At 12 weeks post-surgery, the middle segments of the grafts were isolated and fixed with 4% paraformaldehyde. Cross-sectional slices with a thickness of 10.0 μm were prepared using a cryosection system. These sections were immunostained using the mouse anti-myelin basic protein (MBP) antibody (1:200; Abcam, UK) and rabbit anti-NF200 antibody (1:200; Servicebio, China). Fluorescence images were captured using a fluorescence microscope (H600L, Nikon, Japan), and the regenerated axon area and MBP and NF200 positive area were quantified and merged using Image J 1.8 software.
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