Recombinant Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935)

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 ab236220, the same antibody clone in a different buffer formulation.

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 ab236220, the same antibody clone in a different buffer formulation.

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).

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 21508957).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1: 70 seconds; Lanes 2- 4: 3 minutes.

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).

Exposure time: Lane 1: 3 minutes; Lane 2: 3.25 seconds; Lane 3: 3 minutes

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

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).

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

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).

The molecular weight observed is consistent with what has been described in the literature (PMID: 21508957).

Exposure times: Lane 1: 3 minutes; Lane 2: 37 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

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).

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

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).

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

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).

The upper band around 130 kDa is full length EPHB2 which is consistent to previous report (PMID: 21508957), while the lower band around 80 kDa could be non-specific band or cleaved fragment (PMID: 18713744). The expression on rat brain tissue is consistent with what has been described in the literature (PMID: 16840724).

This blot was developed using a higher sensitivity ECL substrate.

Blocking/Dilution buffer: 5% NFDM/TBST.

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).