Rabbit anti mbp antibody

The expression of the MBP was evaluated by removing the media and then adding a trypsin-EDTA solution (GIBCO, Grand Island, NY, USA) to the plates in order to detach the cells after the cells had grown sufficiently in the groups. The enzyme activity of trypsin was counteracted by subjoining a soybean trypsin inhibitor (Sigma-Aldrich, MO, USA) and centrifuging the cells. First, the cells were fixed with a 2% paraformaldehyde solution for 15 minutes and then they were washed 3 times with phosphate-buffered saline.5 Subsequently, the cells were stained with a diluted rabbit anti-MBP antibody (Abcam, UK, 1:500) overnight in a refrigerator. Afterward, the cells were rewashed and incubated with a diluted Alexa-Fluor 488 goat anti-rabbit secondary antibody (Eugene, OR, USA, 1:1000) for 45 minutes in the dark. Finally, the cells were resuspended in 300 µL of phosphate-buffered saline and flow cytometry was carried out using a FACSCalibur System. The data were collected and analyzed using the FlowJo 7.6 software. According to flowcytometry histograms, the percentage of the positive cells that expressed the MBP was calculated and then the data were entered into the GraphPad Prism 6.01 software and quantitatively compared between the groups.

To obtain accurate data for immunohistochemistry, free-floating sections from all rats were processed carefully under the same conditions as described previously [30 (link)]. For each animal, tissue sections were selected with reference to a rat brain atlas [31 ], between 3.00 and 4.08 mm posterior to the bregma. Ten sections, 90 µm apart, were sequentially treated with 0.3% hydrogen peroxide (H₂O₂) in PBS for 30 min and 10% normal horse serum in 0.05 M PBS for 30 min thereafter. The sections were then incubated with a rabbit anti-MBP antibody (1:1,000, Abcam, Cambridge, UK) overnight at 25℃, before being sequentially treated with either biotinylated goat anti-rabbit IgG, or a streptavidin-peroxidase complex (1:200, Vector, Burlingame, CA). Sections were visualized by the reaction with 3,3-diaminobenzidine tetrachloride (Sigma) in 0.1 M Tris-HCl buffer (pH 7.2). The sections were then dehydrated, before being mounted onto gelatin-coated slides, using Canada balsam (Kanto, Tokyo, Japan).
In order to establish the specificity of primary antibody, procedure included the omission of the anti-MBP antibody, goat anti-rabbit, and substitution of normal goat serum for the anti-MBP antibody. As a result, immunoreactivity disappeared completely in tissues. All experiment procedures in the present study were performed under the same circumstance and in parallel.

HFIP was purchased from Merck, Germany. Polydimethylsiloxane (PDMS) was bought from Dow Corning, USA. PCL (molecular mass of 80 kDa) was from Sigma-Aldrich, USA. Coverslips with smooth surface (φ = 1.2 cm) and bovine serum albumin (BSA) were bought from Feiao Co. Ltd., China. IKVAV peptide and FITC-labeled IKVAV peptide were purchased from GL Biochem, China. PBS (pH 7), AO II, trypsin, and Dulbecco’s modified Eagle’s medium (DMEM) were both purchased from HyClone Co. Ltd. Fetal bovine serum was from Gibco-Invitrogen, Canada. FITC-labeled phalloidin, 4′,6-diamidino-2-phenylindole (DAPI), cell counting kit 8, Triton X-100, PI, polyvinylidene difluoride (PVDF) membranes, and dopamine hydrochloride were all bought from Sigma-Aldrich, USA. GDNF kit, BDNF kit, and NGF kit for ELISA test were all purchased from Boster Co. Ltd., China. BCA kit was bought from Beyotime Co. Ltd., China. Heregulin and forskolin were both purchased from Amresco, USA. Rabbit anti-MBP antibody, rabbit anti-Smad4 antibody, rabbit anti–α-tubulin antibody, and horseradish peroxidase (HRP)–conjugated secondary goat anti-rabbit immunoglobulin G antibody were all bought from Abcam, UK. All other reagents and solvents used in the present study were of reagent grade unless otherwise specified.