Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized BxPC-3 (human pancreas adenocarcinoma epithelial cell, Left) / SK-N-BE(2) (human neuroblastoma neuroblast, Right) cells labelling 68kDa Neurofilament/NF-L (C-term) with ab315814 at 1/500 dilution (0.1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

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

Low expression : BxPC-3.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling 68kDa Neurofilament/NF-L (C-term) with ab315814 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cerebrum (PMID : 34557955).
The section was incubated with ab315814 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-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SK-N-BE(2) (human neuroblastoma neuroblast) cells labelling 68kDa Neurofilament/NF-L (C-term) with ab315814 at 1/500 (2.046 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 SK-N-BE(2) cell line.
Low expreesion : BxPC-3.
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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling 68kDa Neurofilament/NF-L (C-term) with ab315814 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human liver (PMID : 2420946).
The section was incubated with ab315814 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human Alzheimer's disease cerebrum tissue labeling 68kDa Neurofilament/NF-L (C-term) with ab315814 at 1/5000 (0.102 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on cerebrum of human Alzheimer's disease.
The section was incubated with ab315814 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.

• WB

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Western blot - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

Low expression : BxPC-3, ARPE-19.

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

The identity of the higher MW band at approximately 100 kDa is unknown.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] (<a href='/en-us/products/primary-antibodies/68kda-neurofilament-nf-l-c-term-antibody-epr28784-63-ab315814'>ab315814</a>) at 1/1000 dilution

Lane 1:

SK-N-BE(2) (human neuroblastoma neuroblast) whole cell lysate at 50 µg

Lane 2:

BxPC-3 (human pancreas adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 3:

ARPE-19 (human eye epithelial cell) whole cell lysate at 50 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 1/100000 dilution

Observed band size: 70 kDa,124 kDa

false

Exposure time: 59s

• WB

Supplier Data

Western blot - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

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

Negative control : liver (PMID : 2420946).

The expression profile/ molecular weight observed isconsistent with what has been described in the literature (PMID : 33009465).

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

Exposure time : Lane 1 : 1 second, lanes 2-4 : 3 seconds.

All lanes:

Western blot - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] (<a href='/en-us/products/primary-antibodies/68kda-neurofilament-nf-l-c-term-antibody-epr28784-63-ab315814'>ab315814</a>) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

Human spinal cord tissue lysate at 20 µg

Lane 3:

Human alzheimers brain tissue lysate at 20 µg

Lane 4:

Human liver tissue lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 75 kDa,36 kDa

false

• I-ELISA

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Indirect ELISA - Anti-68kDa Neurofilament/NF-L (C-term) antibody [EPR28784-63] - BSA and Azide free (AB315815)

This data was developed using ab315814, the same antibody clone in a different buffer formulation.

Indirect ELISA analysis of ab315814 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution.

Antigen : Human Neurofilament light polypeptide 1,Human Neurofilament light polypeptide 2.

Antigen concentration : 1000 ng/ml