重组Anti-160 kD Neurofilament Medium抗体[EPR23510-76]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized wild-type HEK-293T (human embryonic kidney epithelial cell, Right)/ 160 kD Neurofilament Medium knockout HEK-293T cells (Left) labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 μg). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Positive staining on human wild-type HEK-293T cell line (ab255449), while no staining on human NEFM knockout HEK-293T cells (ab266741).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunofluorescent analysis of parental HEK-293T (EDWT04) and NEFM KO HEK-293T (ED040746) cells labelling 160 kD Neurofilament Medium with ab254348 at 1/50 (8.86 μg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was (Blue).

Confocal image showing cytoplasmic staining in parental HEK-293T cells and no staining in NEFM KO HEK-293T cells.

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on human cerebellum.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on human cerebrum.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (blue).

Negative control : No staining on mouse liver (PMID : 30541916) is observed.

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Negative control : No staining on mouse liver.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of paraffin-embedded mouse cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on mouse cerebellum.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunofluorescent analysis of mouse primary neural/glia cells fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100, labelling 160 kD Neurofilament Medium with ab254348 at 1/50 (8.86 μg/ml) dilution. ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain at 1/500 (4 μg/ml). Followed by secondary ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2 μg/ml) (Green). ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) was used as secondary counterstain at 1/1000 (2 μg/ml) (Red). The nuclear counterstain was DAPI (Blue).

Confocal image showing cytoplasmic staining in mouse primary neuron cells.
Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.

Negative Control 1 : ab150120 1/1000 (2 μg/ml)

Negative Control 2 : ab11267 1/500 (4 μg/ml), secondary : ab150077 1/1000 (2μg/ml)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunofluorescent analysis of rat primary neural/glia cells fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100, labelling 160 kD Neurofilament Medium with ab254348 at 1/50 (8.86 μg/ml) dilution. ab11267 Anti-MAP2 mouse monoclonal antibody was used to counterstain at 1/500 (4 μg/ml). Followed by secondary ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (2 μg/ml) (Green). ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) was used as secondary counterstain at 1/1000 (2 μg/ml) (Red). The nuclear counterstain was DAPI (Blue).

Confocal image showing cytoplasmic staining in rat primary neuron cells.
Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.

Negative Control 1 : ab150120 1/1000 (2 μg/ml)

Negative Control 2 : ab11267 1/500 (4 μg/ml), secondary : ab150077 1/1000 (2 μg/ml)

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized rat primary neuron cells cells labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 μg)/ Right compared with a Rabbit monoclonal IgG isotype control (ab172730) / Left.

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized mouse primary neuron cells labelling 160 kD Neurofilament Medium with ab254348 at 1/500 dilution (0.1 μg)/ Right compared with a Rabbit monoclonal IgG isotype control (ab172730) / Left.

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (blue).

Negative control : No staining on rat liver (PMID : 30541916) is observed.

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

Positive staining on rat cerebrum is observed.

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488 )at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue labelling 160 kD Neurofilament Medium with ab254348 at 1/4000 (0.111 μg/ml) dilution followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The section was incubated with ab254348 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Positive staining on rat cerebellum.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling 160 kD Neurofilament Medium with ab254348 at 1/100 (4.43 μg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (Green). The nuclear counterstain was DAPI (Blue).

Positive staining on mouse cerebrum is observed.

Secondary antibody control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488 )at 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

• IP

Supplier Data

Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

160 kD Neurofilament Medium was immunoprecipitated from 0.35 mg rat brain tissue lysate 10 μg with ab254348 at 1/30 dilution (2 μg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Rat brain tissue lysate 10 μg.

Lane 2 : ab254348 IP in rat brain tissue lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab254348 in rat brain tissue lysate.

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

Exposure time : 5.5 secs.

All lanes:

Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

Predicted band size: 102 kDa

Observed band size: 160 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

160 kD Neurofilament Medium was immunoprecipitated from 0.35 mg mouse brain tissue lysate 10 μg with ab254348 at 1/30 dilution (2 μg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab254348 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Mouse brain tissue lysate 10 μg.

Lane 2 : ab254348 IP in mouse brain tissue lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab254348 in mouse brain tissue lysate.

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

Exposure time : 3.25 secs.

All lanes:

Immunoprecipitation - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348)

Predicted band size: 102 kDa

Observed band size: 160 kDa

false

• ELISA

Supplier Data

ELISA - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

ELISA analysis using ab254348 at a range of 0-1000 ng/ml followed by a Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L）at 1/2500 dilution. Antigen concentration : 1000 ng/ml.

Antigens : Mouse 160 kD Neurofilament Medium.

• WB

Lab

Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

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

The molecular weight is consistent to what has been described in the literature (PMID : 19239416, PMID : 27000625).

Negative control : liver (PMID : 30541916).

Exposure time : 10 seconds.

All lanes:

Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Human nerve tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 102 kDa

Observed band size: 160 kDa

false

• WB

Lab

Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

Blocking and diluting buffer and concentration : Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lanes 1-3 : Merged signal (red and green). Green - ab254348 observed at 160 kDa. Red - loading control ab8245 observed at 36 kDa.

ab254348 Anti-NEFM antibody [EPR23510-76] was shown to specifically react with NEFM in wild-type HEK-293T cells. Loss of signal was observed when the knockout cell line ab266741 (knockout cell lysate ab257103) was used. Wild-type and NEFM knockout samples were subjected to SDS-PAGE. ab254348 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at at 4°C overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

NEFM knockout HEK-293T whole cell lysate at 20 µg

Lane 3:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 102 kDa

Observed band size: 160 kDa

false

• WB

Lab

Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (AB254348)

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

The molecular weight is consistent to what has been described in the literature (PMID : 19239416, PMID : 27000625).

Negative control : liver (PMID : 30541916).

Exposure times : Lane 1-3 : 3.25 secs; Lane 4-6 : 10 secs.

All lanes:

Western blot - Anti-160 kD Neurofilament Medium antibody [EPR23510-76] - Neuronal Marker (ab254348) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse cerebellum tissue lysate at 20 µg

Lane 3:

Mouse liver tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Rat cerebellum tissue lysate at 20 µg

Lane 6:

Rat liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 102 kDa

Observed band size: 160 kDa

false