Anti-EpCAM antibody

6 product images

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  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 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, Goat polyclonal Secondary Antibody to Mouse at 1/1000 dilution (shown in pseudocolour red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

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  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

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  Western blot - Anti-EpCAM antibody (ab71916)

  Secondary antibody - goat anti-rabbit HRP (H&L preadsorbed; Goat Anti-Rabbit IgG H&L (HRP) 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 (Goat Anti-Rabbit IgG H&L (HRP) preadsorbed ab97080) at 1/5000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 35 kDa

  Observed band size: 35 kDa, 40 kDa

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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

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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

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  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).