Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Immunohistochemical analysis of paraffin-embedded Human liver sections labelling 160 kD Neurofilament Medium with ab92539 at 0.105μg/ml, followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) system. Counterstained with Hematoxylin.

Negative control : no staining on human liver. The section was incubated with ab92539 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methanol permeabilized SH-SY5Y (human neuroblastoma epithelial cell) cells labelling Neurofilament with ab92539 at 1/20 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (Red).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Immunohistochemical analysis of paraffin-embedded Human astrocytoma sections labelling 160 kD Neurofilament Medium with ab92539 at 0.105μg/ml, followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) system. Counterstained with Hematoxylin.

Positive staining on human astrocytoma. The section was incubated with ab92539 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. 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-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling 160 kD Neurofilament Medium with ab92539 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cerebrum. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Immunohistochemical analysis of paraffin-embedded Human glioblastoma multiforme tissue labeling 160 kD Neurofilament Medium with ab92539 at 1/1000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human glioblastoma multiforme. The primary antibody was incubated for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Immunofluorescent analysis of 100% Methanol-fixed, 0.1% TritonX-100 permeabilized SH-SY5Y cells labelling Neurofilament with ab92539 at 1/200 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody at 1/1000 dilution, was co-incubated with ab92539 overnight at 4° C, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at 1/1000 dilution at RT for 45 min (Magenta).

Confocal image showing cytoplasmic staining in SH-SY5Y cells (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Immunohistochemistry image of Neurofilament 160 kDa staining in a section of frozen normal human cerebral cortex*.

The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab92539 at 1/500 dilution. The secondary antibody was ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• WB

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Western blot - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

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

All lanes:

Western blot - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (ab92539) at 1/10000 dilution

All lanes:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 15 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

Predicted band size: 160 kDa

true

Exposure time: 3s

• WB

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Western blot - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (AB92539)

Anti-NEFM antibody [EP2460] (ab92539) staining at 0.01μg/ml, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab92539 was shown to bind specifically to NEFM. A band was observed at 140-160 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in NEFM knockout cell line. To generate this image, wild-type and NEFM knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-160 kD Neurofilament Medium antibody [EP2460] - Neuronal Marker (ab92539) at 0.01 µg/mL

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

NEFM knockout HEK-293T cell lysate at 20 µg

Lane 3:

SK-N-BE cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Lane 5:

SH-SY5Y cell lysate at 20 µg

Secondary

Lanes 1 - 5:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 5:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 102 kDa

Observed band size: 140-160 kDa

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