Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Fluorescence multiplex immunohistochemical analysis of the human jejunum (Formalin/PFA-fixed paraffin-embedded sections). Panel A : merged staining of anti-Muc2 (ab272692, magenta; Opal™690), anti-Eph receptor B2 (ab252935, green; Opal™520) and anti-ErbB3 / HER3 (ab236220, red; Opal™570) on human jejunum. Panel B : anti-Muc2 stained on goblet cells. Panel C : anti-Eph receptor B2 stained on intestinal stem and progenitor cells. Panel D : anti-ErbB3 / HER3 stained on epithelial cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The section was incubated in three rounds of staining : in the order of ab272692 at 1/2000 dilution (0.252 μg/ml), ab252935 at 1/1000 dilution  (0.527 μg/ml), and ab236220 at 1/3000 dilution (0.75 μg/ml) 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections). Panel A : merged staining of anti-Muc2 (ab272692, magenta; Opal™690), anti-Eph receptor B2 (ab252935, green; Opal™520) and anti-ErbB3 / HER3 (ab236220, red; Opal™570) on human colon. Panel B : anti-Muc2 stained on goblet cells. Panel C : anti-Eph receptor B2 stained on intestinal stem and progenitor cells. Panel D : anti-ErbB3 / HER3 stained on epithelial cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody. The section was incubated in three rounds of staining : in the order of ab272692 at 1/2000 dilution (0.252 μg/ml), ab252935 at 1/1000 dilution  (0.527 μg/ml), and ab236220 at 1/3000 dilution (0.75 μg/ml) 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using ab252935, the same antibody clone in a different buffer formulation.

Immunohistochemistry analysis of Formalin/PFA-fixed paraffin-embedded sections rat small intestine tissue labelling Sodium/Hydrogen Exchanger 3 with ab307365 at 1 : 5000 dilution (B), MUC2 with ab272692 at 1 : 2000 dilution (C) Eph receptor B2 with ab252935 at 1 : 500 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-Sodium/Hydrogen Exchanger 3 (green; Opal™520), anti-MUC2 (magenta; Opal™690) and anti-Eph receptor B2 (gray; Opal™570) on rat small intestine.

Panel B : anti-Sodium/Hydrogen Exchanger 3 staining apical and luminal facing edges of surface cells in rat small intestine.

Panel C : anti-MUC2 staining goblet cells in rat small intestine.

Panel D : anti-Eph receptor B2 staining membrane of stem cells in rat small intestine.

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

The section was incubated in three rounds of staining : in the order of ab307365, ab272692 and ab252935 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using ab252935, the same antibody clone in a different buffer formulation.

Immunohistochemistry analysis of (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse colon tissue labelling Eph receptor B2 with ab252935 at 1/500 dilution (B), GSDMC2 + GSDMC3 with ab229896 at 1/500 dilution (C) and Serotonin with ab315150 at 1/500 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-Eph receptor B2 (green; Opal™520), anti-GSDMC2/3 (magenta; Opal™690) and anti-Serotonin (gray; Opal™570) on mouse stomach.

Panel B : anti-Eph receptor B2 staining stem cells in mouse stomach.

Panel C : anti-GSDMC2/3 staining epithelium in mouse stomach.

Panel D : anti-Serotonin staining enterochromaffin cells in mouse stomach.

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

The section was incubated in three rounds of staining : in the order of ab252935, ab229896 and ab315150 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using ab252935, the same antibody clone in a different buffer formulation.

Immunohistochemistry analysis of Formalin/PFA-fixed paraffin-embedded sections mouse colon tissue labelling Sodium/Hydrogen Exchanger 3 with ab307365 at 1 : 5000 dilution (B), MUC2 with ab272692 at 1 : 2000 dilution (C) Eph receptor B2 with ab252935 at 1 : 500 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-Sodium/Hydrogen Exchanger 3 (green; Opal™520), anti-MUC2 (magenta; Opal™690) and anti-Eph receptor B2 (gray; Opal™570) on mouse colon.

Panel B : anti-Sodium/Hydrogen Exchanger 3 staining apical and luminal facing edges of surface cells in mouse colon.

Panel C : anti-MUC2 staining goblet cells in mouse colon.

Panel D : anti-Eph receptor B2 staining membrane of stem cells in mouse colon.

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

The section was incubated in three rounds of staining : in the order of ab307365, ab272692 and ab252935 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using ab252935, the same antibody clone in a different buffer formulation.

Immunohistochemistry analysis of Formalin/PFA-fixed paraffin-embedded sections rat colon tissue labelling Sodium/Hydrogen Exchanger 3 with ab307365 at 1 : 5000 dilution (B), MUC2 with ab272692 at 1 : 2000 dilution (C) Eph receptor B2 with ab252935 at 1 : 500 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-Sodium/Hydrogen Exchanger 3 (green; Opal™520), anti-MUC2 (magenta; Opal™690) and anti-Eph receptor B2 (gray; Opal™570) on rat colon.

Panel B : anti-Sodium/Hydrogen Exchanger 3 staining apical and luminal facing edges of surface cells in rat colon.

Panel C : anti-MUC2 staining goblet cells in rat colon.

Panel D : anti-Eph receptor B2 staining membrane of stem cells in rat colon.

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

The section was incubated in three rounds of staining : in the order of ab307365, ab272692 and ab252935 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on rat colon (PMID : 24151532) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver tissue labeling Eph receptor B2 with ab252935 at 1/50 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. No staining in rat liver. Negative control : liver (PMID : 19366806). Counterstained with DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using ab252935, the same antibody clone in a different buffer formulation.

Immunohistochemistry analysis of Formalin/PFA-fixed paraffin-embedded sections mouse small intestine tissue labelling Sodium/Hydrogen Exchanger 3 with ab307365 at 1 : 5000 dilution (B), MUC2 with ab272692 at 1 : 2000 dilution (C) Eph receptor B2 with ab252935 at 1 : 500 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-Sodium/Hydrogen Exchanger 3 (green; Opal™520), anti-MUC2 (magenta; Opal™690) and anti-Eph receptor B2 (gray; Opal™570) on mouse small intestine.

Panel B : anti-Sodium/Hydrogen Exchanger 3 staining apical and luminal facing edges of surface cells in mouse small intestine.

Panel C : anti-MUC2 staining goblet cells in mouse small intestine.

Panel D : anti-Eph receptor B2 staining membrane of stem cells in mouse small intestine.

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

The section was incubated in three rounds of staining : in the order of ab307365, ab272692 and ab252935 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using ab252935, the same antibody clone in a different buffer formulation.

Immunohistochemistry analysis of (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse colon tissue labelling Eph receptor B2 with ab252935 at 1/500 dilution (B), GSDMC2 + GSDMC3 with ab229896 at 1/500 dilution (C) and Trefoil Factor 3 with ab300427 at 1/500 dilution (D). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Panel A : merged staining of anti-Eph receptor B2 (green; Opal™520), anti-GSDMC2/3 (magenta; Opal™690) and anti-Trefoil Factor 3 (gray; Opal™570) on mouse colon.

Panel B : anti-Eph receptor B2 staining stem cells in mouse colon.

Panel C : anti-GSDMC2/3 staining epithelium in mouse colon.

Panel D : anti-Trefoil Factor 3 staining goblet cells in mouse colon.

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

The section was incubated in three rounds of staining : in the order of ab252935, ab229896 and ab300427 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using ab252935, the same antibody clone in a different buffer formulation.

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse small intestine tissue staining Eph receptor B2 with ab252935 at 1/500 dilution, ab314897 anti-CaMKII beta used at 1/5000 dilution and ab238123 anti-MPTX used at a 1/2000 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

Panel A : merged staining of anti-Eph receptor B2 (green; Opal™520), anti-CaMKII beta (magenta; Opal™690) and anti-MPTX (gray; Opal™570) on mouse small intestine.
Panel B : anti-Eph receptor B2 staining membrane of stem cells in mouse small intestine.
Panel C : anti-CaMKII beta staining endocrine cells in mouse small intestine.
Panel D : anti-MPTX staining membrane of Paneth cells in mouse small intestine.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab259935, ab314897 and ab238123 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• WB

Lab

Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

This data was developed using the same antibody clone in a different buffer formulation (ab252935 ).

Western blot : Anti-EPHB2 antibody [EPR22427-268] (ab252935) staining at 1/3000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab252935 was shown to bind specifically to EPHB2. A band was observed at 130 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EPHB2 knockout cell line. To generate this image, wild-type and EPHB2 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Eph receptor B2 antibody [EPR22427-268] (<a href='/en-us/products/primary-antibodies/eph-receptor-b2-antibody-epr22427-268-ab252935'>ab252935</a>) at 1/3000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

EPHB2 knockout HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human EPHB2 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ephb2-knockout-hct116-cell-line-ab287395'>ab287395</a>)

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

HepG2 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 130 kDa

false

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse colon tissue labeling Eph receptor B2 with ab252935 at 1/250 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on the membrane of epithelial cells, especially located in the basal crypt region (PMID : 19621336) is observed. Counterstained with DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human bladder cancer (PMID : 24151532, 25148033) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver tissue labeling Eph receptor B2 with ab252935 at 1/50 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. No staining in mouse liver. Negative control : liver (PMID : 19366806). Counterstained with DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on mouse colon (PMID : 24151532) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on the base of the crypts of human colon (PMID : 24151532) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-Fr

Unknown

Immunohistochemistry (Frozen sections) - Anti-Eph receptor B2 antibody [EPR22427-268] - BSA and Azide free (AB255274)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat colon tissue labeling Eph receptor B2 with ab252935 at 1/250 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on the membrane of epithelial cells, especially located in the basal crypt region (PMID : 19621336) is observed. Counterstained with DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab252935).