Anti-EZHIP antibody [EPR28396-24]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-EZHIP antibody [EPR28396-24] (AB316856)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (human embryonic kidney epithelial cell, Left) / U-2 OS (human bone osteosarcoma epithelial cell, Right) cells labelling EZHIP with ab316856 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 : 293T.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EZHIP antibody [EPR28396-24] (AB316856)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling EZHIP with ab316856 at 1/500 (0.996 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human placenta (PMID : 27699219).
The section was incubated with ab316856 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-EZHIP antibody [EPR28396-24] (AB316856)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-2 OS (human bone osteosarcoma epithelial cell) cells labelling EZHIP with ab316856 at 1/100 (4.98 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 U-2 OS cell line.
Low expression : 293T (PMID : 34880314).
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.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-EZHIP antibody [EPR28396-24] (AB316856)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human placenta tissue staining GCSF Receptor with ab324569 at a 1/100 dilution, ab316856 anti-EZHIP used at 1/500 dilution and ab324497 anti-CLEC14A used at a 1/1000 dilution.

Panel A : anti-GCSF Receptor (green; Opal™520), anti-EZHIP (magenta; Opal™690) and anti-CLEC14A (gray; Opal™570) on human placenta.

Panel B : anti-GCSF Receptor staining cytoplasm of trophoblast in human placenta.

Panel C : anti-EZHIP staining nucleus in human placenta.

Panel D : anti-CLEC14A staining endothelium in human placenta.

Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab324569, ab316856 and ab324497 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

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-EZHIP antibody [EPR28396-24] (AB316856)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling EZHIP with ab316856 at 1/500 (0.996 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on human kidney (PMID : 31451685).
The section was incubated with ab316856 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-EZHIP antibody [EPR28396-24] (AB316856)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling EZHIP with ab316856 at 1/500 (0.996 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : No staining on human liver.
The section was incubated with ab316856 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-EZHIP antibody [EPR28396-24] (AB316856)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling EZHIP with ab316856 at 1/500 (0.996 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human testis (PMID : 31451685).
The section was incubated with ab316856 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

• IP

Supplier Data

Immunoprecipitation - Anti-EZHIP antibody [EPR28396-24] (AB316856)

EZHIP was immunoprecipitated from 0.35 mg U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate with ab316856 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316856 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate
Lane 2 : ab316856 IP in U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316856 in U-2 OS whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-EZHIP antibody [EPR28396-24] (ab316856) at 1/30 dilution

All lanes:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-EZHIP antibody [EPR28396-24] (AB316856)

Low expression : 293T (PMID : 34880314).

The molecular weight observed is consistent with what has been described in the literature (PMID : 34880314).

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

All lanes:

Western blot - Anti-EZHIP antibody [EPR28396-24] (ab316856) at 1/1000 dilution

Lane 1:

U-2OS (human bone osteosarcoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell 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: 40 kDa,75 kDa,36 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-EZHIP antibody [EPR28396-24] (AB316856)

Negative control : A204.

The molecular weight observed is consistent with what has been described in the literature (PMID : 34880314).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

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

All lanes:

Western blot - Anti-EZHIP antibody [EPR28396-24] (ab316856) at 1/1000 dilution

Lane 1:

U-2OS (human bone osteosarcoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

A-204 (human muscle rhabdomyosarcoma 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 1/20000 dilution

Observed band size: 40 kDa,75 kDa,36 kDa

false

Exposure time: 10s