Anti-CLEC14A antibody [EPR30281-548]

• mIHC

Lab

Multiplex immunohistochemistry - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

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-CLEC14A antibody [EPR30281-548] (AB324497)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling CLEC14A with ab324497 at 1/1000 (0.498 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on endothelial cells of human colon carcinoma.
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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

Immunohistochemical analysis of paraffin-embedded (A) HUVEC (human umbilical vein endothelial cell) cell pellet. (B) HEK-293 (human embryonic kidney epithelial cell) cell pellet. tissue labeling CLEC14A with ab324497 at 1/1000 (0.498 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) HUVEC cell pellet, no staining on (B) HEK-293 cell pellet.
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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

Immunohistochemical analysis of paraffin-embedded Human clear cell carcinoma of kidney tissue labeling CLEC14A with ab324497 at 1/1000 (0.498 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on endothelial cells of human clear cell carcinoma of kidney.
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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling CLEC14A with ab324497 at 1/1000 (0.498 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on endothelial cells of 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling CLEC14A with ab324497 at 1/1000 (0.498 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on endothelial cells of human placenta.
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

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

Flow cytometric analysis of HEK-293 (human embryonic kidney epithelial cell, Left) / HUVEC (human umbilical vein endothelial cell, Right) cells labelling CLEC14A with ab324497 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).

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

Gated on viable cells.
Low expression : HEK-293.

• WB

Supplier Data

Western blot - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

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

In lane 1, the lysate was stored at -80℃ prior to Western Blotting. The bands beneath the target band (60 kDa) are likey to be degradation products. In lane 2, To minimize protein degradation, cell was lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28671670).

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

All lanes:

Western blot - Anti-CLEC14A antibody [EPR30281-548] (ab324497) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell) whole cell lysate (Frozen lysate) at 20 µg

Lane 2:

HUVEC (human umbilical vein endothelial cell) whole cell lysate (Fresh 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: 40-60 kDa,36 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

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

Low expression : HEK-293, THP-1.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28671670).

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

All lanes:

Western blot - Anti-CLEC14A antibody [EPR30281-548] (ab324497) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 2:

EA.hy926 (human somatic cell hybrid endothelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

THP-1 (human monocytic leukemia monocyte) 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: 40-60 kDa,100 kDa,36 kDa

false

Exposure time: 92s

• WB

Supplier Data

Western blot - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

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

CLEC14A is a glycoprotein of approximately 40-60, 100 kDa and detected as a 35-50, 90 kDa band after treated with Peptide : N-glycosidase F (PNGase F).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28671670).

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

All lanes:

Western blot - Anti-CLEC14A antibody [EPR30281-548] (ab324497) at 1/1000 dilution

Lane 1:

Untreated HUVEC whole cell lysate at 20 µg

Lane 2:

HUVEC whole cell lysate treated with Peptide: N-glycosidase F (PNGase F) 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: 40-60 kDa,100 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CLEC14A antibody [EPR30281-548] (AB324497)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 28671670).

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

All lanes:

Western blot - Anti-CLEC14A antibody [EPR30281-548] (ab324497) at 1/1000 dilution

Lane 1:

Human hypothalamus tissue lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human colon tissue lysate at 20 µg

Lane 4:

Human small intestine tissue lysate at 20 µg

Lane 5:

Human pancreas tissue lysate at 20 µg

Lane 6:

Human spleen 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: 40-60 kDa,100 kDa,36 kDa

false

Exposure time: 180s