Mouse brain tissue sections and cells were incubated with following specific antibodies: anti‐LC3B (PM036, MBL), anti‐Iba‐1 (019‐19741, Wako), anti‐NeuN (MAB377, Millipore), anti‐P62 (ab56416, Abcam), anti‐LAMP2 (sc‐18822), anti‐ki67 (ab15580, Abcam), anti‐MAP2 (ab11267, Abcam), anti‐MBP (ab62631, Abcam), and anti‐Iba1 (019‐19741, Wako Pure Chemical Industries, OSA) in a humidified container for 12 h at 4°C, followed by incubation with fluorescent conjugated secondary antibodies (Santa Cruz Biotechnology).
Ab62631
Manufactured by Abcam
Sourced in United States
Ab62631 is a monoclonal antibody that recognizes the protein GAPDH. It is a commonly used antibody in western blotting and immunohistochemistry applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab6319, by Abcam (3 mentions)
Mab377, by Merck Group (2 mentions)
Anti neun, by Merck Group (2 mentions)
Ab89780, by Abcam (2 mentions)
Ab85615, by Abcam (2 mentions)
Ab203126, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ecl kit, by Boster Bio (2 mentions)
Sc 33666, by Santa Cruz Biotechnology (2 mentions)
Ab155279, by Abcam (2 mentions)
Ab11267, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab56416, by Abcam (1 mentions)
Gb21301, by Wuhan Servicebio Technology (1 mentions)
Gb25301, by Wuhan Servicebio Technology (1 mentions)
Caseviewer, by 3DHISTECH (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab15690, by Abcam (1 mentions)
Ab10062, by Abcam (1 mentions)
Fv1200, by Olympus (1 mentions)
11 protocols using ab62631
1
Immunohistochemistry of Mouse Brain Tissue
2
Spinal Cord Tissue Immunohistochemistry
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The wax‐embedded spinal tissue in each group (n = 5) was sectioned into slices at a thickness of 4 μm. After wax removal, hydration and antigen repair, the section was rinsed with phosphate‐buffered saline (PBS, pH = 7.4) three times for 5 minutes each time. Then, the section was subjected to blocking using 10% bovine serum albumin (BSA) for 30 minutes. The primary antibodies anti‐NeuN (MAB377; Millipore) and anti‐myelin basic protein (anti‐MBP, ab62631, Abcam) were added and incubated overnight at 4°C. After washing with PBS three times for 5 minutes each time, the secondary antibody (goat anti‐mouse, GB21301; goat anti‐mouse, GB25301; Servicebio) was incubated at room temperature for 50 minutes. Then, DAPI staining, antiquenching and sealing were conducted. The fluorescence images were scanned with a Pannoramic 250 (Pannoramic MIDI; 3D HISTECH); then, the images were viewed with CaseViewer (3D HISTECH) software. Photoshop CC (Adobe) and Image‐Pro Plus 6.0 software (Media Cybernetics) were used to process and analyse the images. The integrated optical density (IOD) of MBP in the whole cross‐sectional image was quantified, the NeuN‐positive cells in the anterior horns of the spinal cord were manually counted, and the counts were averaged. Both tests were quantified in a double‐blind manner.
3
Immunofluorescence Staining of Rat Brain
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Samples were collected from animals at 24 h after perforation for double-fluorescence staining, performed as previously described [18 (link)]. The steps of cells immunofluorescence refer to the experimental method reported by Elliot H Choi et al. [23 (link)]. In brief, rat brains were perfused with 0.9 % saline first, and next the 4% paraformaldehyde, then fixed with 4% paraformaldehyde, dehydrated, and frozen at −80 °C. The samples were then sliced into 10 μm thick frozen sections, which were exposed O/N at 4 °C to the individual primary antibodies as follows: anti-CNPase (1:50, Abcam, ab6319), anti-Iba-1 (1:50, Abcam, ab15690), anti-GFAP (1:50, Abcam, ab10062), anti-myelin basic protein (MBP, 1:50, Abcam, ab62631), anti-Dexras1 (1:50, Abcam, ab78459), anti-NG2 (1:50, Biorbyt, orb382135), anti-NEUN (1:50, Merck Millipore, Burlington, MA, USA). The appropriate secondary antibody (Proteintech, SA00003-1/SA00009-2, Wuhan, China) was incubated with the brain sections at RT for 2 h, and the sections were observed and photographed using a fluorescence microscope (FV1200, Olympus, Tokyo, Japan). The main brain region we looked at was the subcortical area of the left cerebral hemisphere that was enriched for Dexras1 after subarachnoid hemorrhage.
4
Immunostaining and Imaging of Myelination in Co-cultured DRGs and Schwann Cells
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Co-cultured DRGs and Schwann cells and crushed proximal nerve stumps were collected and processed as previously described (Zanazzi et al., 2001). For immunostaining, cells and tissue slices were fixed with 4% paraformaldehyde/PBS for 5 minutes at room temperature, blocked with 5% goat serum for 30 minutes at room temperature and incubated with primary antibodies: anti-myelin basic protein (MBP) (1:500; AB62631, Abcam St Louis, MO, USA), anti-peripheral myelin protein zero (P0) (1:125; AB31851, Abcam), or anti-myelin-associated glycoprotein (MAG) (1:200; AB89780, Abcam) overnight at 4°C. Specimens were then incubated with Cy3-conjugated goat anti-rabbit secondary antibody (1:1,000; Sigma) for 2 hours at room temperature, and were then mounted on microscope slides. Samples were visualized and images captured using an optical and epifluorescence microscope (Axio Imager M2, Carl Zeiss Microscopy GmbH, Jena, Germany). All assays were performed in triplicate.
5
Mouse Brain Immunolabeling and Imaging
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Mice were perfused with 4% PFA, sliced on a cryostat at 40 μm, and immunolabeled with rabbit anti‐NeuN (1:500, #24307; Cell Signaling), rabbit anti‐PV (1:500, #ab11427; Abcam), rat anti‐CTIP2 (1:500, #ab18465; Abcam), mouse anti‐MBP (1:500, #ab62631; Abcam), and secondary antibodies were Invitrogen, 1:500, as previously described.21 Confocal images were taken on Nikon Eclipse Ti‐U at 10x/0.45 NA or 20x/0.95 NA, 1024 × 1024 or 512 × 512 frame size, 5 μm z‐interval. Myelinated axons were imaged on Leica SP8 STED at 93x/1.3 NA, glycerol. GFP FLEX callosum fibers were imaged on CSU‐W1 SoRa Yokogawa Spinning Disk Confocal, Nikon at 100x/1.35 NA, silicon, or 60x/1.49 NA, oil, 2304 × 2304, 0.5 μm step size. Excitation lasers were 405, 488, and 561. Imaris version 9.8 (Bitplane) was used for visualization and Adobe Photoshop CC for figure display.
We injected AAV9 CamKII0.4.eGFP.WPRE.rBG (#105541‐AAV9; Addgene), Cre‐driven AAV9 pCAG‐FLEX‐EGFP‐WPRE (#51502‐AAV9; Addgene), or green retrograde Lumafluor RetroBeads IX in the right premotor cortex of TRAP2 or C57Bl/6 mice (AP +2.6 mm, ML −1.8 mm, DV −0.5 mm; 100 nl) or in the right VL (anterior VL: AP −1.05 mm, posterior VL: AP: −1.40 mm, ML −1.00 mm, DV −3.70 mm) with Hamilton syringe (#7634‐01) and allowed 2 weeks for the viral expression.
We injected AAV9 CamKII0.4.eGFP.WPRE.rBG (#105541‐AAV9; Addgene), Cre‐driven AAV9 pCAG‐FLEX‐EGFP‐WPRE (#51502‐AAV9; Addgene), or green retrograde Lumafluor RetroBeads IX in the right premotor cortex of TRAP2 or C57Bl/6 mice (AP +2.6 mm, ML −1.8 mm, DV −0.5 mm; 100 nl) or in the right VL (anterior VL: AP −1.05 mm, posterior VL: AP: −1.40 mm, ML −1.00 mm, DV −3.70 mm) with Hamilton syringe (#7634‐01) and allowed 2 weeks for the viral expression.
6
Tissue Clarification and Immunohistochemistry Protocol
7
Quantifying Oligodendrocyte Lineage Cells
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Mice were anesthetized and then perfused, tissues were cryopreserved, embedded, and sectioned as described above. Immunohistochemistry was performed with primary antibodies against PDGFα (1:500; AF1062, R&D Systems), CC1 (1:500; OP80, EMD Millipore), BrdU (1:500; ab6326, Abcam), MBP (1:1000; ab62631, Abcam), and Olig2 (1:500; ab109186, Abcam). The stained sections were visualized by laser scanning confocal microscopy (Nikon Eclipse Ti microscope, NIS-Elements AR Imaging). Quantification of CC1+/Olig2+/BrdU+ and PDGFα+/Olig2+ cells was performed using the count quantification function of a NIS-Elements AR analysis system. The MBP+ areas were quantified using the defined area method of the NIS-Elements AR analysis system (for details, see the supplementary material).
8
Quantification of Target Proteins by Immunoblotting
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The levels of the target proteins were measured with the following primary antibodies: anti-CNPase (1:1,000, ab6319, Abcam), anti-myelin basic protein (MBP; 1:1,000, ab62631, Abcam), anti-neuron glia 2 (NG2; 1:1,000, sc-33666, SANTA CRUZ), anti-GSK3β (1:1,000, D5C5Z, CST), anti-phosphorylated (Ser9) GSK3β (p-ser9-GSK3β; 1:1,000, D85E12, CST), anti-5-HT1AR (1:1,000, ab85615, Abcam), anti-human gene and protein symbol ACTB/ACTB (β-actin; 1:5,000, ab8226, Abcam), and anti-heat-shock protein 90 (HSP90; 1:2,000, ab203126, Abcam). The corresponding secondary antibodies (goat anti-rabbit IgG, CW0103 and goat anti-rabbit IgG, CW0102, CWBIO, P. R. China) for each protein were applied at a dilution of 1:1,000, followed by development with an ECL kit (AR1111, Boster, P. R. China). The band intensity was quantified using Image Lab software (version 5.2.1).
9
Mitochondrial Dynamics in Peripheral Nerves
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Proteins from sciatic nerves or cells were separated using RIPA and centrifuged at 12,000 g for 15 min at 4°C. Proteins were denatured at 95°C for 5 min and analyzed on SDS-PAGE, transfer to the PVDF membrane, and immunoblotting for Mfn2 (1:1000, ab124773, Abcam), MPB (1:1000, ab62631, Abcam), Sox10 (1:1000, ab155279, Abcam), Phospho-Drp1(Ser616) (1:1000, 4494, CST), OPA1 (1: 1000, ab157457, Abcam), Mfn1 (1:500, OM209507, omnimabs), Drp1 (1:1000, ab184247, Abcam), and β-actin (1:2000, TA-09, ZSGB) was performed overnight at 4°C.
10
Immunohistochemical Analysis of HO-1, MSR1, and MBP
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