Anti-Nogo A + Nogo D antibody [EPR26286-56]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Mouse primary neuron cells labelling Nogo A + Nogo D with ab315792 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control.

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse liver (fresh frozen) tissue labeling Nogo A + Nogo D with ab315792 at 1/500 (1.004 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Negative control : confocal image showing no staining on mouse liver. The section was incubated with ab315792 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Nogo A + Nogo D with ab315792 at 1/4000 (0.126 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on mouse liver.
The section was incubated with ab315792 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Immunohistochemical analysis of paraffin-embedded Mouse spinal cord tissue labeling Nogo A + Nogo D with ab315792 at 1/4000 (0.126 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse spinal cord (PMID : 15751227).
The section was incubated with ab315792 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling Nogo A + Nogo D with ab315792 at 1/4000 (0.126 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum (PMID : 17356385).
The section was incubated with ab315792 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background.

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cell cells labelling Nogo A + Nogo D with ab315792 at 1/1000 (0.502 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing cytoplasmic staining in mouse primary neural/glia cell.
Confocal scanning Z step was set as 0.3 ?m followed by image processing with maximum Z projection.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain at 1/500 (4ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (fresh frozen) tissue labeling Nogo A + Nogo D with ab315792 at 1/500 (1.004 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Confocal image showing positive staining on mouse cerebrum. The nuclear counterstain was DAPI (Blue). The section was incubated with ab315792 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse splenocytes cells labelling Nogo A + Nogo D with ab315792 at 1/1000 (0.502 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/mL) dilution (Green).

Negative control : Confocal image showing no staining in mouse splenocytes (PMID : 11978832).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution.

• WB

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Western blot - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : liver (PMID : 11978832).

The molecular weight observed is consistent with what has been described in the literature (PMID : 27786289).

The bands between 50 kDa and 200 kDa are likely to be degraded target fragments.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Nogo A + Nogo D antibody [EPR26286-56] (ab315792) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 50 µg

Lane 2:

Mouse spinal cord tissue lysate at 50 µg

Lane 3:

Mouse liver tissue lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 50 kDa,200 kDa,36 kDa

false

Exposure time: 15s

• WB

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Western blot - Anti-Nogo A + Nogo D antibody [EPR26286-56] (AB315792)

Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Nogo A + Nogo D antibody [EPR26286-56] (ab315792) at 1/1000 dilution

All lanes:

Mouse testis tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 200 kDa

false

Exposure time: 70s