Cultured cells or brain slices (30 μm) were fixed with 4% paraformaldehyde followed by three washes in PBS and permeabilized with 0.1% Triton X-100 for 10 min (except for O4, O1 immunostainning) then blocked in 10% goat serum (Invitrogen Corp. USA) for 1 h following incubation with the primary antibody overnight at 4 °C. Primary antibodies were diluted in blocking solutions as follows: mouse anti-MBP (Biolegend, USA; 1:500), mouse anti-APC, CC1 clone (Millipore, USA; 1:500), mouse anti-O4 (R&D Systems Inc, USA, 1:800), mouse anti-O1 (R&D Systems Inc, USA, 1:800), rabbit anti-PDGFRα (Cell Signalling Technology, USA; 1:500), rabbit anti-glial fibrillary acidic protein (GFAP, Biolegend, USA; 1:500) mouse anti-BrdU (Sigma St Louis, USA; 1:500). The tissue or cells were then washed with PBS and incubate in Alexa Flour-conjugated secondary antibodies (Invitrogen Corp. USA; 1:500) for 1 h. Images were obtained using Olympus photomicroscope and analyzed using Image-Pro plus 6.0 software.
Mouse anti mbp
Manufactured by BioLegend
Sourced in United States
Mouse anti-MBP is a laboratory reagent used for the detection and analysis of myelin basic protein (MBP) in various experimental settings. It functions as a specific antibody that binds to MBP, enabling researchers to study the expression and distribution of this protein in biological samples.
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Lab products found in correlation
Mouse anti gapdh, by Thermo Fisher Scientific (2 mentions)
Rabbit anti glut3, by Abcam (2 mentions)
Rabbit anti glut1, by Abcam (2 mentions)
Blotting grade blotter, by Bio-Rad (2 mentions)
Spectramax m3 plate reader, by Molecular Devices (2 mentions)
Mouse anti plp, by Merck Group (2 mentions)
Mouse anti cnpase, by Merck Group (2 mentions)
Rabbit anti mct2, by Merck Group (2 mentions)
Mouse anti mog, by Merck Group (2 mentions)
Rabbit anti connexin 43, by Merck Group (2 mentions)
Mouse anti mag, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Photomicroscope, by Olympus (1 mentions)
Alexa flour conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Rabbit anti pdgfrα, by Cell Signaling Technology (1 mentions)
Mouse anti o1, by R&D Systems (1 mentions)
Mouse anti o4, by R&D Systems (1 mentions)
Glial fibrillary acidic protein (gfap), by BioLegend (1 mentions)
6 protocols using mouse anti mbp
1
Immunohistochemistry of Cultured Cells and Brain Slices
2
Quantification of MBP and Actin in Injured Brains
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At P10 and P17, the HI injured brains were carefully removed as completed and rapidly as possible. The tissue was homogenized by a magnetic grinder in protein extract solution (T-PER Tissue Protein Extraction Reagent, Halt Protease Inhibitor Cocktail; Thermo Fisher Scientific, Waltham, MA, USA). The supernatants were reserved after centrifugation. Supernatant with 50 µg of total protein was then denatured at 95 ℃ for 5 min and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (12%). Next, the protein was transferred to a nitrocellulose membrane and saturated for 2 h with blocking buffer (7% defatted milk) followed by the primary antibody (mouse anti-MBP, 1:1,000, BioLegend; mouse anti-actin, 1:1,000, Origene Technologies, Rockville, MD, USA) and incubation overnight at 4 ℃. After rewarming for 30 min, the membranes were incubated in the secondary antibody [goat anti-mouse IgG-horseradish peroxidase (HRP), 1:500; goat anti-rabbit IgG-HRP, 1:500, Absin, Shanghai, China] for 1 h. A ChemiDocXRS+ imaging system and ImageLab software (Bio-Rad, Hercules, CA, USA) were used for visualization and densitometric analysis after 5 min of immersion in enhanced chemiluminescence substrate (Thermo Fisher Scientific). The strips were analyzed using ImageLab version 5.0 (Bio-Rad).
3
Oligodendrocyte Precursor Cell Differentiation
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OPCs were cultured on 10 mm glass coverslips for 2 days. To analyze differentiation, cells were cultured for an additional 5 days in transferred astrocyte medium with or without LIF-neutralizing antibody (10 μg; R&D Systems, catalog no. AB-449-NA) or normal goat IgG (10 μg; R&D Systems, catalog no. AB-108-C). Cells were fixed in 4% paraformaldehyde and blocked with PBS containing 3% bovine serum albumin and 0.1% Triton X-100 for 10 min. Cells were incubated at 4°C overnight with primary antibodies against PDGFRα (rabbit anti-PDGFRα, 1:1000; Santa Cruz Biotechnology, catalog no. sc-338) and MBP (mouse anti-MBP, 1:500; BioLegend, catalog no. 836504). Cells were then labeled with the secondary antibodies Alexa Fluor donkey anti-rabbit (594) and anti-mouse (488) IgG (1:300; Invitrogen) at room temperature for 1.5 hours in the dark. After staining nuclei with 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount-G (Southern Biotechnology Associates), fluorescence images were acquired using the FluoView FV10i confocal microscope (Olympus).
4
Western Blot Analysis of Protein Expression
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The protein concentration of each sample was measured using the Lowry method and the colorimetric reaction was measured on a SpectraMax M3 plate reader (Molecular Devices). 10–40µg of total protein was loaded on mini Protean TGX Precast gels (Biorad). After electrophoresis, proteins were blotted on a PVDF membrane (Biorad) and blocked in 5% Blotting Grade Blotter (Biorad) diluted in Tris buffered saline (TBS) containing 0.1% Tween 20 (TBST). Blots were then incubated with either of the following antibodies: chicken anti-human MCT1 antibody, mouse anti-PLP (MilliporeSigma), mouse anti CNPase (MilliporeSigma), rabbit anti-MCT2 (MilliporeSigma), rabbit anti-GLUT1 (abcam), rabbit anti-GLUT3 (abcam), mouse anti-MBP (Biolegend), mouse anti-MOG (MilliporeSigma), rabbit anti-Connexin-43 (MilliporeSigma), mouse anti-GAPDH (Thermo Scientific), and mouse anti-MAG (MilliporeSigma)(for more details see Key Resources Table ). Membranes were overnight incubated with primary antibodies at 4°C. Membranes were washed with TBST and incubated with HRP labeled secondary antibodies for 1 hour at room temperature. After two washes in TBST and one wash in TBS, membranes were exposed to ECL reagent for 2 min. (Thermo Scientific) and chemiluminescent signal was detected with the LAS Imager 4000 (GE Healthcare).
5
Western Blot Analysis of Protein Expression
6
Western Blot for Notch Signaling
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Cells were harvest in lysis buffer on ice. Sample proteins were loaded on SDS-PAGE gel and blotted onto PVDF membranes. After blocking in 5% (w/v) non-fat milk for 1 h, proteins were probed with primary antibodies at 4 °C overnight. Primary antibodies were diluted in 5% bovine serum albumin blocking buffer as follows: rabbit anti-Jagged1 (Cell Signalling Technology, USA; 1:500), rabbit anti-Notch1 (Cell Signalling Technology, USA; 1:1000), mouse anti-MBP (Biolegend, USA; 1:1000). Membranes were then incubated in horseradish peroxidase-conjugated secondary antibody for 1 h, the immunoreactive proteins can be detected by chemiluminescence ECL agents (Millipore, USA).
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