Anti-MNDA antibody [EPR28267-44]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MNDA antibody [EPR28267-44] (AB316967)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labelling MNDA with ab316967 at 1/500 dilution (0.1ug)/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.

Cells were stained with anti-CD14 conjugated to Alexa Fluor®647. Fixed with 2% PFA for 10 min followed by intracellularly staining with rabbit IgG or EPR28267-44

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MNDA antibody [EPR28267-44] (AB316967)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1(human monocytic leukemia monocyte) cells labelling MNDA with ab316967 at 1/50 (10.2 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 nuclear staining in THP-1 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : K-562 (PMID : 12112016).
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 (2.5 ug/mL) dilution (Magenta). 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MNDA antibody [EPR28267-44] (AB316967)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (human monocytic leukemia monocyte) / right and K-562 (human chronic myelogenous leukemia lymphoblast) / left cells labelling MNDA with ab316967 at 1/500 dilution (0.1ug)/(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.

Negative control : K-562 (PMID : 12112016)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MNDA antibody [EPR28267-44] (AB316967)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling MNDA with ab316967 at 1/100 (5.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : Scattered staining of immune cells in the human liver.
The section was incubated with ab316967 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-MNDA antibody [EPR28267-44] (AB316967)

Immunohistochemical analysis of paraffin-embedded Human hodgkin's lymphoma tissue labeling MNDA with ab316967 at 1/100 (5.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human hodgkin's lymphoma.
The section was incubated with ab316967 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-MNDA antibody [EPR28267-44] (AB316967)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling MNDA with ab316967 at 1/100 (5.1 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human tonsil.
The section was incubated with ab316967 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-MNDA antibody [EPR28267-44] (AB316967)

Negative control : Jurkat

Low expression : liver

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

Exposure time : Lane1-2 : 15seconds, lane3-5 : 180 seconds

All lanes:

Western blot - Anti-MNDA antibody [EPR28267-44] (ab316967) at 1/1000 dilution

Lane 1:

U-937 (human histiocytic lymphoma monocyte) whole cell lysate at 20 µg

Lane 2:

Jurkat (human t cell leukemia t lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 3:

Human spleen tissue lysate at 20 µg

Lane 4:

Human tonsil tissue lysate at 20 µg

Lane 5:

Human liver 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 1/100000 dilution

Observed band size: 27 kDa,15 kDa

false

• WB

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Western blot - Anti-MNDA antibody [EPR28267-44] (AB316967)

Negative control : K-562(PMID : 12112016)

The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

MNDA (full-length 55 kDa), cleaved fragments (27 kDa) are observed

The molecular weight observed is consistent with what has been described in the literature(PMID : 20395555)

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

All lanes:

Western blot - Anti-MNDA antibody [EPR28267-44] (ab316967) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 2:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell 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 1/100000 dilution

Observed band size: 27 kDa,55 kDa,36 kDa

false

Exposure time: 37s