Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Immunohistochemical analysis of paraffin-embedded Human cerebrum labeling Myelin Basic Protein with ab7349 at 1/4000 dilution (0.259 μg/ml) followed by a ready to use Goat Anti-Rat IgG H&L (HRP polymer; ab214882). The section was incubated with ab7349 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Immunohistochemical analysis of paraffin-embedded Human cerebrum labeling Myelin Basic Protein with ab7349 at 1/4000 dilution (0.259 μg/ml) followed by a ready to use Goat Anti-Rat IgG H&L (HRP polymer; ab214882). The section was incubated with ab7349 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with DAPI.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized mouse primary neural/glia cells labelling Myelin Basic Protein with ab7349 at a 1/200 dilution, followed by Goat anti-Rat (Alexa Fluor^® 488) (ab150157) secondary antibody at a 1/1000 dilution. Confocal image showing positive staining in mouse primary oligodendroglia cells (shown in green). Nuclear DNA was labeled with DAPI (shown in blue).

Counterstained with Anti-MAP2 antibody (ab183830) at a 1/1000 dilution, followed by Goat Anti-Rabbit secondary (Alexa Fluor^® 594) (ab150080) at a 1/1000 dilution (shown in magenta).

Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

The negative controls are as follows :

-ve control 1 : Myelin Basic Protein (ab7349) at a 1/200 dilution, followed by Goat anti-Rabbit (Alexa Fluor^® 594) (ab150080) at a 1/1000 dilution.

-ve control 2 : Anti-MAP2 (ab183830) at a 1/1000 dilution, followed by Goat anti-Rat (Alexa Fluor^® 488) (ab150157) at a 1/1000 dilution.

• WB

Supplier Data

Western blot - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

The molecular weight observed was in consistency with the literature (PMID : 9299539).

Blocking buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (ab7349) at 1/5000 dilution

All lanes:

Human cerebellum tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 18.5 kDa

true

Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

The molecular weight observed was in consistency with the literature (PMID : 9299539).

Blocking buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 3 seconds; Lane 2 : 1 second.

All lanes:

Western blot - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (ab7349) at 1/5000 dilution

Lane 1:

Mouse brain tissue lysate at 10 µg

Lane 2:

Rat brain tissue lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 14-21.5 kDa

false

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Immunocytochemistry-immunofluorescence using Anti-Myelin Basic Protein antibody [12], ab7349. Publication image from Rawji, K. S. et al., 2020, Acta Neuropathol, 32030468. Legend direct from paper.

Myelin debris activates microglia and myelin phagocytosis requires the scavenger receptor CD36. a Representative images of mouse microglia exposed to myelin debris and fluorescently conjugated latex beads (red) and stained for Iba1 (green) and MBP (red). b A greater percentage of microglia phagocytose myelin debris compared to latex beads over time. c Representative images of neonate, young adult, and aged microglia exposed to myelin debris and stained for Iba1 (green) and MBP (red). d Neonate and young microglia phagocytose myelin debris with a significantly greater efficiency than aged microglia. e Representative images of mouse microglia exposed to myelin debris and stained for CD11b (green) and iNOS (red). f Myelin debris stimulates the expression of iNOS in CD11b+ microglia. g Log2 fold change of RNA expression of pro- and anti-inflammatory genes from microglia activated with myelin debris for 2 h compared to naïve controls. h Venn diagram representation of the association of scavenger receptors in the set of differentially expressed genes (DAA) whose expression in microglia was regulated upon activation and that is affected due to aging. i, j Heat maps of expression levels of scavenger receptors (in DAA) in microglia isolated from young and aging mice and activated for 2 (i) and 12 h (j). Euclidean distance matrix was used for hierarchical clustering. Difference in expression of scavenger receptors in microglia from aged mice was compared to microglia from young mice and the estimated log2 fold change was plotted. k Representative images of mouse microglia exposed to myelin debris and treated with a CD36 receptor antagonist (sulfo-N-succinimidyl oleate, SSO) and stained for Iba1 (red) and MBP (green). l SSO reduces microglia phagocytosis of myelin debris. m Representative images of young and aged microglia exposed to myelin debris and treated with control, lipofectamine (Lipo), and overexpression of CD36 (CD36 OE) and stained for Iba1 (red) and MBP (green). n Overexpression of CD36 significantly enhances myelin phagocytosis by young and aged mouse microglia as well as human microglia. Values are represented as mean with the standard error of the mean. Results were analyzed with a two-way repeated-measures ANOVA with a Bonferroni’s post hoc test (b, d), a one-way repeated-measures ANOVA with a Dunnett’s post hoc test (f, l), a Wald test and Benjamin–Hochberg p value adjustment (g, i, j), a two-sided Chi-square test with Yates’ correction (h), and a one-way ANOVA with a Bonferroni’s post hoc test (n each cell type analyzed individually). Values are indicative of triplicate cultures. For panels a, c, e, k, m, scale bars equal 50 µm. For panels b, d, f, l, n, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. For panels g, i, j, *p < 0.05

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Immunocytochemistry-immunofluorescence using Anti-Myelin Basic Protein antibody [12], ab7349. Publication image from Rawji, K. S. et al., 2020, Acta Neuropathol, 32030468. Legend direct from paper.

Myelin debris activates microglia and myelin phagocytosis requires the scavenger receptor CD36. a Representative images of mouse microglia exposed to myelin debris and fluorescently conjugated latex beads (red) and stained for Iba1 (green) and MBP (red). b A greater percentage of microglia phagocytose myelin debris compared to latex beads over time. c Representative images of neonate, young adult, and aged microglia exposed to myelin debris and stained for Iba1 (green) and MBP (red). d Neonate and young microglia phagocytose myelin debris with a significantly greater efficiency than aged microglia. e Representative images of mouse microglia exposed to myelin debris and stained for CD11b (green) and iNOS (red). f Myelin debris stimulates the expression of iNOS in CD11b+ microglia. g Log2 fold change of RNA expression of pro- and anti-inflammatory genes from microglia activated with myelin debris for 2 h compared to naïve controls. h Venn diagram representation of the association of scavenger receptors in the set of differentially expressed genes (DAA) whose expression in microglia was regulated upon activation and that is affected due to aging. i, j Heat maps of expression levels of scavenger receptors (in DAA) in microglia isolated from young and aging mice and activated for 2 (i) and 12 h (j). Euclidean distance matrix was used for hierarchical clustering. Difference in expression of scavenger receptors in microglia from aged mice was compared to microglia from young mice and the estimated log2 fold change was plotted. k Representative images of mouse microglia exposed to myelin debris and treated with a CD36 receptor antagonist (sulfo-N-succinimidyl oleate, SSO) and stained for Iba1 (red) and MBP (green). l SSO reduces microglia phagocytosis of myelin debris. m Representative images of young and aged microglia exposed to myelin debris and treated with control, lipofectamine (Lipo), and overexpression of CD36 (CD36 OE) and stained for Iba1 (red) and MBP (green). n Overexpression of CD36 significantly enhances myelin phagocytosis by young and aged mouse microglia as well as human microglia. Values are represented as mean with the standard error of the mean. Results were analyzed with a two-way repeated-measures ANOVA with a Bonferroni’s post hoc test (b, d), a one-way repeated-measures ANOVA with a Dunnett’s post hoc test (f, l), a Wald test and Benjamin–Hochberg p value adjustment (g, i, j), a two-sided Chi-square test with Yates’ correction (h), and a one-way ANOVA with a Bonferroni’s post hoc test (n each cell type analyzed individually). Values are indicative of triplicate cultures. For panels a, c, e, k, m, scale bars equal 50 µm. For panels b, d, f, l, n, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. For panels g, i, j, *p < 0.05

• IF

CiteAb

Immunofluorescence - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Immunofluorescence using Anti-Myelin Basic Protein antibody [12], ab7349. Publication image from Zaremba, A. et al., 2015, Nat Commun, 25766071. Legend direct from paper.

Guanabenz decreases IFN-γ-induced hypomyelination in rat cerebellar slice cultures.(a–e) Anti-MBP staining of myelinated fibres (a, arrowheads) in slice cultures that were (a) untreated, (b) treated with IFN-γ or (c–e) concomitantly treated with IFN-γ and 2.5, 5.0 or 10.0 µM guanabenz. Images representative of two or three slices per treatment; the experiment was performed twice. (f) Electron microscopy analysis of slice cultures. Note the significant increase in the number of myelinated axons (arrowheads) when guanabenz and IFN-γ were concomitantly added to slices. Myelinated axons per field were determined by analysis of a minimum of 200 axons per condition. (g) Toluidine blue staining of slices left untreated, treated with IFN-γ alone or treated with IFN-γ and guanabenz. Images represent a minimum of three sections per treatment. Unpaired t-test, *P<0.05. Data are presented as mean±s.e.m. Scale bars, 100 µm (a–e), 2 µm (f) and 20 µm (g). WM : white matter, GL : granule cell layer, PCL : Purkinje cell layer.

• IHC

CiteAb

Immunohistochemistry - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Immunohistochemistry using Anti-Myelin Basic Protein antibody [12], ab7349. Publication image from Romero-Ramirez, L. et al., 2020, J Biomed Sci, 32066435. Legend direct from paper.

The presence of non-phosphorylated filaments as an indicator of axonal damage. Ten weeks after SCI immune staining with Smi32 antibody (red) was combined with myelin basic protein-IR (green) and Hoechst-33342 nuclear staining (blue). a-f Overview of transverse spinal cord sections at intervals of approximately 3.2 mm from 8 mm anterior to 8 mm posterior of the lesion site; 5x objective, scale bar in a. Note the presence of Smi32-binding in the ascending dorsal columns anterior but not posterior of the lesion site and in white matter tracts in all sections. g Non-phosphorylated neurofilament in ascending fiber tracts anterior of the site of injury, 20x objective. h-i Higher magnification of Smi32-IR in white matter (h) and motor neurons in the ventral horn (i), 40x objective, scale bar in i. No Smi32 staining was observed in the white matter of animals without SCI (see Fig. 8)

• WB

CiteAb

Western blot - Anti-Myelin Basic Protein antibody [12] - Oligodendrocyte Marker (AB7349)

Western Blotting using Anti-Myelin Basic Protein antibody [12], ab7349. Publication image from Monk, K. R. et al., 2015, Nat Commun, 25607655. Legend direct from paper.

Loss of GPR56 causes CNS hypomyelination in mice.(a) Reduced FluoroMyelin staining was observed in the CC of P14 and P28 brains of Gpr56−/− mice (lower panel) compared with their littermate control (upper panel). Scale bar, 500 µm. (b) Bar graphs depicted percentage of area myelinated. Myelin is reduced at P14 (*P=0.0144; unpaired t-test, n=4 per genotype) and P28 (*P=0.0499; unpaired t-test, n=3 per genotype) in the CC of Gpr56−/− compared with controls. (c) Western blot analyses of MBP and PLP expression in the CC of Gpr56+/− and Gpr56−/− mice at P7-28. Loading control : β-actin. (d,e) Bar graphs depicted relative optical density of MBP and PLP to the loading control β-actin. MBP protein was significantly reduced in the CC of Gpr56−/− compared with Gpr56+/− littermates on P14 (*P=0.0285), P21 (*P=0.0315) and P28 (*P=0.0443). Unpaired t-test, n=3 per genotype. PLP protein was significantly decreased in the CC of Gpr56−/− compared with Gpr56+/− littermates on P7 (**P=0.0091), P14 (***P<0.0001), P21 (**P=0.0011) and P28 (**P=0.003). Unpaired t-test, n=4 per genotype. Error bars are means ± s.e.m.

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