For preparation of tissue sections, mouse cerebrum and cerebellum were harvested and fixed in 4% paraformaldehyde for 24 h at 4 °C. Tissues were then equilibrated in 30% sucrose/PBS for 48 h. Sagittal or coronal sections (30 μm) were cut using a freezing sledge microtome. Free-floating sections were processed for immunofluorescence as previously described [19 (link)]. Sections were labeled with an antibody against PLP (Abcam #ab28486, 1/200) in three mice per group. Nissl stains and Purkinje cell counts were performed in two mice per group as previously described [20 (link)]. Imaging was performed using a Zeiss Axiovert M2 microscope.
Ab28486
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab28486 is a monoclonal antibody specific for the human CXorf26 protein. CXorf26 is a protein of unknown function located on the X chromosome. The antibody is suitable for use in various immunoassay applications to detect and study the CXorf26 protein.
Automatically generated - may contain errors
Lab products found in correlation
Ab9610, by Merck Group (2 mentions)
Axiovert m2 microscope, by Zeiss (1 mentions)
Donkey anti mouse af647, by Thermo Fisher Scientific (1 mentions)
Ab15580, by Abcam (1 mentions)
Ab7817, by Abcam (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Ab214666, by Abcam (1 mentions)
Quantity one version 4, by Bio-Rad (1 mentions)
Prism 8, by GraphPad (1 mentions)
Ab53041, by Abcam (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
Fluorochrome conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ab5320, by Merck Group (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
β actin 8457, by Cell Signaling Technology (1 mentions)
Ab76472, by Abcam (1 mentions)
Sc 57351, by Santa Cruz Biotechnology (1 mentions)
Ab7795, by Abcam (1 mentions)
Ab89780, by Abcam (1 mentions)
Ab40390, by Abcam (1 mentions)
14 protocols using ab28486
1
Immunohistochemical Analysis of Mouse Brain
2
Immunofluorescence Analysis of Aortic Tissue
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Slides of aortic tissue were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized in 0.1% PBS-Triton for 30 min. The samples were blocked in 5% BSA/PBS/0.1% Triton-X 100 for 1 h and then incubated with the primary antibodies anti-Kruppel-like factor 4 (Klf4, ab214666), anti-neural cell adhesion molecule (Ncam1, ab28486), anti-Ki67 (ab15580), anti-alpha-smooth muscle actin (ab7817) (all from Abcam, USA), and anti-vitronectin (Vtn, MA5-24083, Invitrogen, USA). After several PBS washes, the slides were incubated for 2 h at room temperature with the secondary antibodies AF647 donkey anti-mouse and AF568 donkey anti-rabbit (Invitrogen, USA). Immunofluorescence was assessed using a Zeiss LSM 880 confocal laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany). The fluorescence values were measured with ImageJ software (developed at the National Institutes of Health). At least 100 cells were analyzed for each group.
3
Quantifying Myelin Protein Expression
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G72Tg and WT mouse hippocampal cytoplasmic Mog, Cldn11, Plp protein expression (n = 10 per group) was assessed using anti-Mog (ab28766, Abcam, Cambridge, UK, 1:15,000 dilution, 10 μg loaded per sample), anti-Cldn11 (Osp) (ab53041, Abcam, 1:5000 dilution, 2 μg loaded per sample) and anti-Plp (ab28486, Abcam, 1:5000 dilution, 2 μg loaded per sample) primary antibodies, respectively. Western blot was performed as previously described [12 (link)]. Transfer membranes were stained with Coomassie Brilliant Blue R-250 (BioRad Laboratories, Hercules, CA, USA) and signal intensities were compared to ensure equal protein loading. For signal intensity quantification QuantityOne (version 4.4.0, BioRad Laboratories, Hercules, CA, USA) was used. G72Tg and WT group differences in Western blots were assessed by the non-parametric, Mann–Whitney test using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Results were considered statistically significant for p < 0.05.
4
Immunofluorescence Quantification of Oligodendrocyte Lineage Cells
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Dissociated PAR1+/+ or PAR1−/− NSCs at passage 3 were plated as monolayers in triplicate on poly-L-lysine coated glass cover slips and grown for 2 DIV in media containing EGF and bFGF or for 5 DIV after growth factor withdrawal. At each end point, cultures were fixed with 2% paraformaldehyde and stained for NG2 (AB5320, EMD Millipore), Olig2 (AB9610, EMD Millipore), or PLP (ab28486, Abcam) using immunofluorescence techniques. Species appropriate fluorochrome conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Sections were cover slipped with VECTASHIELD Hardset containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA). Five 20X microscopic fields encompassing the center and 4 poles of each coverslip were digitally imaged using an Olympus BX51 microscope (Olympus, Center Valley, PA). The mean number of NG2+ or Olig2+ cells was enumerated without knowledge of genotype and expressed as a ratio of the total number of DAPI+ cells counted in the same fields.
5
Comprehensive Antibody Validation Protocol
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Antibodies against Gbp2 (ab203238), GFAP (ab53554, ab7260), LCN2 (ab63929), LRP1 (ab92544), MAG (ab89780), MBP (ab40390), NF200 (ab7795), PLP (ab28486) and S100A10 (ab76472) were purchased from Abcam, UK; antibody against CD31 (550274) was purchased from BD Biosciences, USA; antibodies against GFAP (3670S), GFP (2955S, 2956S), p38 (9212S), pp38 (4511S) and β-actin (8457S) were purchased from Cell Signaling Technology, USA; antibodies against CC1 (OP80), dMBP (AB5864) and Olig2 (AB9610) were purchased from Millipore, USA; antibodies against C3d (AF2655), Galectin3 (AF1197), LAMP1 (AF4320) and LCN2 (AF1857) were purchased from R&D Systems, USA; antibodies against Gbp2 (sc-166960) and LRP1 (sc-57351) were purchased from Santa Cruz Biotechnology, USA; antibody against pLRP1 (PA5-101013) was purchased from Thermo Fisher, USA; antibody against Iba1 (019-19741) was purchased from Wako, Japan. All antibodies were used at a dilution of 1:50 to 1:2000 for immunofluorescence and 1:800 to 1:5000 for immunoblotting according to the manufacturer’s instructions. Alexa Fluor 488, 594 and 647 conjugated secondary antibodies were purchased from Jackson, USA.
6
Immunohistochemical Analysis of Neural Cell Types
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Immunohistochemistry was carried out to investigate the morphological features of neural cells, the numbers of each cell type, and their glycosylation status. The primary antibodies used were as follows: anti-beta III tubulin (ab78078; Abcam, Cambridge, USA), anti-160 kDa NF medium (ab7794; Abcam), anti-MBP (ab626931; Abcam), anti-MAG (9043S CST, Danvers, USA), anti-MOG (ab32760; Abcam), anti-PLP (ab28486; Abcam), anti-APC (OP80; Merck), anti-Tbr1 (ab31940; Abcam), and fluorescein isothiocyanate (FITC)-conjugated WFA (Vector Laboratories, Burlingame, USA). The sections were incubated with primary antibodies (1/200 dilution) overnight at 4°C, rinsed three times in PBS, and incubated with Alexa Fluor-488 or Alexa Fluor-546 secondary antibodies (1/200 dilution) for 2 h at room temperature. The stained sections were sealed using cover slips and ProLong Diamond antifade mountant with 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, USA). Immunofluorescence signals were observed and evaluated under a fluorescence microscope (BZ-X710, Keyence, Osaka, Japan). Representative immunohistochemistry images from 2–3 different mice per genotype are shown in Figs 4 , 5 , 6 , 7 and 8 .
7
Western Blot Analysis of Proteolipid Protein
8
Quantification of Myelin Proteins in Optic Nerves
9
Immunocytochemistry of Neural Cell Markers
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Cells cultured on glass coverslips were fixed in 10% formalin (Sigma-Aldrich), permeabilised with 0.3% (v/v) Triton™ X-100 (Sigma-Aldrich) in PBS for 10 min at room temperature, washed with 0.1% (v/v) TWEEN® 20 in PBS, and blocked in 10% (v/v) normal horse serum in 0.1% (v/v) Triton™ X-100 in PBS for 1 h at room temperature. Primary antibodies (1:250 mouse anti-β-III tubulin (Merck Millipore, MAB1637), 2 µg/mL mouse anti-NF-M (Invitrogen, 130700), 11.6 µg/mL rabbit anti-GFAP (Dako, 0334), 1:250 rabbit anti-PLP (Abcam, ab28486), or mouse and rabbit isotype controls (Mesenchymal Stem Cell Laboratory research group, The University of Adelaide, Australia)) were diluted in blocking solution and cells were incubated at 4 °C overnight. Primary antibodies were detected by incubating with secondary antibodies (2 µg/mL donkey anti-mouse IgG Cyanin3 (Merck Millipore, AP192C) or 3 µg/mL donkey anti-rabbit IgG Cyanin3 (Jackson Immunoresearch, 711165152)) for 2.5 h at 4 °C. Coverslips were mounted on microscope slides with ProLong™ Gold Antifade Mountant with DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen).
10
Immunofluorescence Staining of Myelination Markers
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Fentanyl was obtained from Humanwell, Yichang, China; the primary antibodies against mbp (A1664), NG2 (A3592) mog (A5353) were purchased from AB-clonal company (Wuhan, China); the primary antibody against plp1 (ab28486) was purchased from Abcam; the primary antibodies against mag (ABP53248) and mobp (ABP59303) were purchased from Abbkine company (Wuhan, China); cleaved-caspase 3 antibody (#9661) and mbp antibody (#83683) was purchased from Cell Signaling Technology (USA); and the antibodies against GAPDH and horseradish peroxidase (HRP)-linked goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Boster company (Wuhan, China). Color-coded prestained protein marker (10-180 kDa) was purchased from Abbkine company (Wuhan, China). IF-Kine green donkey anti-mouse lgG (A24211) and IF-Kine red donkey anti-rabbit lgG (A24421) were purchased from Abbkine company (Wuhan, China); and z-DEVDfmk was purchased from Selleck (Shanghai, China).
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