Anti-EpCAM antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-EpCAM antibody (AB71916)

ab71916 staining EpCam in HT29 cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab71916 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150120, Goat polyclonal Secondary Antibody to Mouse at 1/1000 dilution (shown in pseudocolour red) and ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody (AB71916)

ab71916 (1 : 160) staining EpCAM in paraffin-embedded human breast tissue, using an automated system (Ventana Discovery).
Using this protocol there is strong membrane staining of the basolateral membranes of normal breast epithelial cells of breast ducts and lobular acini. There is associated weak to moderate staining of the cytoplasm of these cells.

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Standard Retrieval programme. Slides were blocked in 3% H₂O₂ / 4 min / 37°C and incubated with ab71916 (1 : 160 dilution / 2 hours / 37°C). Sections then blocked (4mins / 37°C) and incubated with Dako swine anti-rabbit antibody (1 : 50, 28 min / 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC system (16 min / 37°C) and Ventana DAB map reagent (8 min / 37°C). Slides were counterstained with Haematoxylin and coverslipped in DPX.

For manua

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody (AB71916)

ab71916 (1 : 40) staining EpCAM in paraffin-embedded human breast carcinoma (Grade 2 Invasive Ductal Carcinoma) using an automated system (Ventana Discovery).
Using this protocol there is moderate to strong membrane staining in carcinoma cells which may be apical or complete instead of basolateral. There is associated moderate to strong staining of the cytoplasm of these cells.

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Standard Retrieval programme. Slides were blocked in 3% H₂O₂ / 4 min / 37°C and incubated with ab71916 (1 : 40 dilution / 1 hour / 37°C). Sections then blocked (4mins / 37°C) and incubated with Dako swine anti-rabbit antibody (1 : 50, 28 min / 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC system (16 min / 37°C) and Ventana DAB map reagent (8 min / 37°C). Slides were counterstained with Haematoxylin and coverslipped in D

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody (AB71916)

IHC image of EpCAM staining in human breast adenocarcinoma formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab75962, 1μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EpCAM antibody (AB71916)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon adenocarcinoma labelling EpCAM with ab71916 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

Anti-EpCAM antibody ab71916 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• WB

Unknown

Western blot - Anti-EpCAM antibody (AB71916)

Secondary antibody - goat anti-rabbit HRP (H&L preadsorbed; ab97080)

All lanes:

Western blot - Anti-EpCAM antibody (ab71916) at 1 µg/mL

Lane 1:

Colon (Mouse) Tissue Lysate at 10 µg

Lane 2:

Colon (Rat) Tissue Lysate at 10 µg

Lane 3:

HCT 116 (Human Colorectal Carcinoma) Whole Cell Lysate at 10 µg

Lane 4:

SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate at 10 µg

Lane 5:

Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 6:

HuES7 (Human embryonic stem cell line) Whole Cell Lysate at 10 µg

Lane 7:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa,40 kDa

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• WB

CiteAb

Western blot - Anti-EpCAM antibody (AB71916)

Western Blotting using Anti-EpCAM antibody, ab71916. Publication image from Sengmanivong, L. et al., 2017, Nat Commun, 28084299. Legend direct from paper.

EpCAM silencing leads to unusual tight junction positioning defects at tricellular junctions.(a) Western blot analysis of the EpCAM expression level in control (Caco2 shNT) or EpCAM-deprived (Caco2 shEpCAM #1 and Caco2 shEpCAM #2) clones. GAPDH was used as loading control. (b,c) Confocal microscopy analysis of E-cadherin (b) and Scribble (c) on the apical or lateral sides in control and EpCAM-depleted cells. Scale bars, 5 µm. Nuclei were detected with Hoechst 33342 staining (blue). (d,e) 3D-SIM microscopy analysis of E-cadherin localization in control (d) and EpCAM-deprived (e) cells. Scale bars, 2 µm. (f) Cell shape analysis after E-cadherin immunostaining in control or EpCAM-deprived cells. Scale bars, 5 µm. (g), Statistical analysis of polygonal shape in control or EpCAM-deprived cells. Three independent experiments were carried out. N (Caco2shNT)=517 cells, N (Caco2shEpCAM#1)=550 cells, N (Caco2shEpCAM#2)=427 cells. Caco2 shNT cells with two cell edges=0%, three cell edges=0.455±0.787%, four cell edges=8.673±3.406%, five cell edges =48.154±11.123%, six cell edges=35.652±8.888%, more than six cell edges=7.067±3.851 cells%. Caco2 shEpCAM#1 cells with two cell edges=1.822±0.733 cells%, three cell edges=22.031±5.906%, four cell edges=46.061±3.851%, five cell edges=24.728±4.344%, six cell edges=5.027±2.210%, more than six cell edges=0.330±0.572%. Caco2 shEpCAM#2 cells with two cell edges=0.966±0.848%, three cell edges=12.542±5.942%, four cell edges=46.739±8.082%, five cell edges=32.319±4.777%, six cell edges=6.098±3.412%, more than six cell edges=1.336±1.261%. Unpaired t-tests, *P<0,0001. Values are mean±s.e.m. (h–j) Confocal microscopy analysis of claudin-7, ZO-1 and occludin on the apical or lateral sides in control and EpCAM-depleted cells. (k) Confocal analysis of occludin (red) location at TCs in EpCAM-deprived cells, 3D rendering of z-stacks. Tilted bottom view is presented. Scale bars, 5 µm. Nuclei were detected with Hoechst 33342 staining (blue).

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