Anti-Robo4 antibody [EPR26282-3]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Robo4 with ab300046 at 1/100 (3.77 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on endothelial cells in human colon. The section was incubated with ab300046 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat pancreas tissue staining Robo4 with ab300046 at a 1/100 (3.75 μg/ml) dilution, ab325233 anti-Trypsin used at a 1/500 (0.98 μg/ml) dilution and ab254259 anti-Amylin used at a 1/2000 (0.26 μg/ml) dilution.

Panel A : anti-Robo4 (green; Opal™570), anti-Trypsin (magenta; Opal™690), anti-Amylin (gray; Opal™570) on rat pancreas.

Panel B : anti-Robo4 staining endothelium in rat pancreas.

Panel C : anti-Trypsin staining exocrine gland in rat pancreas.

Panel D : anti-Amylin staining islet cells in rat pancreas.

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

The section was incubated in three rounds of staining : in the order of ab300046, ab325233 and ab254259 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.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse pancreas tissue staining Robo4 with ab300046 at a 1/100 (3.75 μg/ml) dilution, ab325233 anti-Trypsin used at a 1/500 (0.98 μg/ml) dilution and ab254259 anti-Amylin used at a 1/2000 (0.26 μg/ml) dilution.

Panel A : anti-Robo4 (green; Opal™570), anti-Trypsin (magenta; Opal™690), anti-Amylin (gray; Opal™570) on mouse pancreas.

Panel B : anti-Robo4 staining endothelium in mouse pancreas.

Panel C : anti-Trypsin staining exocrine gland in mouse pancreas.

Panel D : anti-Amylin staining islet cells in mouse pancreas.

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

The section was incubated in three rounds of staining : in the order of ab300046, ab325233 and ab254259 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-Robo4 antibody [EPR26282-3] (AB300046)

Immunohistochemical analysis of paraffin-embedded Mouse pancreatic cancer tissue labeling Robo4 with ab300046 at 1/100 (3.77 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on endothelial cells in mouse pancreatic cancer. The section was incubated with ab300046 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 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-Robo4 antibody [EPR26282-3] (AB300046)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Robo4 with ab300046 at 1/100 (3.77 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on endothelial cells in mouse colon. The section was incubated with ab300046 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 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-Robo4 antibody [EPR26282-3] (AB300046)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling Robo4 with ab300046 at 1/100 (3.77 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used. Positive staining on endothelial cells in rat cerebrum. The section was incubated with ab300046 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 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-Robo4 antibody [EPR26282-3] (AB300046)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling Robo4 with ab300046 at 1/100 (3.77 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used. Positive staining on endothelial cells in rat cerebrum. The section was incubated with ab300046 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• WB

Unknown

Western blot - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Blocking / Diluting buffer and concentration : 5% NFDM/TBST

The molecular weight observed is consistent with what have been described in literature (PMID : 27882935).

Negative controls : HeLa, HCT 116(PMID : 11944987, PMID : 11076864 ).

All lanes:

Western blot - Anti-Robo4 antibody [EPR26282-3] (ab300046) at 1/1000 dilution

Lane 1:

Human heart tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Mouse kidney tissue lysate at 20 µg

Lane 4:

Rat spleen tissue lysate at 20 µg

Lane 5:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>);Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG. Secondary ab dilution 1:2000;1:20,000

Predicted band size: 107 kDa

Observed band size: 60 kDa

false

Exposure time: 15s

• WB

Unknown

Western blot - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Blocking / Diluting buffer and concentration :

5% NFDM/TBST

All lanes:

Western blot - Anti-Robo4 antibody [EPR26282-3] (ab300046) at 1/1000 dilution

Lane 1:

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

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HCT 116 (human colorectal carcinoma epithelial cell) 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 1/20000 dilution

Predicted band size: 107 kDa

Observed band size: 60 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Blocking / Diluting buffer and concentration : 5% NFDM/TBST Negative controls : HeLa, HCT 116 (PMID : 11944987, PMID : 11076864).

All lanes:

Western blot - Anti-Robo4 antibody [EPR26282-3] (ab300046) at 1/1000 dilution

Lane 1:

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

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HCT 116 (human colorectal carcinoma epithelial cell) 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 1/20000 dilution

Predicted band size: 107 kDa

Observed band size: 60 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Blocking / Diluting buffer and concentration : 5% NFDM/TBST The molecular weight observed is consistent with what have been described in literature (PMID : 27882935). Negative controls : HeLa, HCT 116(PMID : 11944987, PMID : 11076864 ).

All lanes:

Western blot - Anti-Robo4 antibody [EPR26282-3] (ab300046) at 1/1000 dilution

Lane 1:

Human heart tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

Mouse kidney tissue lysate at 20 µg

Lane 4:

Rat spleen tissue lysate at 20 µg

Lane 5:

Rat kidney tissue lysate at 20 µg

Secondary

Lanes 1 - 5:

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

Lanes 1 - 5:

Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>);Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 107 kDa

Observed band size: 60 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Robo4 antibody [EPR26282-3] (AB300046)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling Robo4 with ab300046 at 1/100 (3.77 µg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on endothelial cells in mouse colon. The section was incubated with ab300046 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.