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 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

All lanes : Anti-EpCAM antibody (ab71916) at 1 µg/ml

Lane 1 : Colon (Mouse) Tissue Lysate
Lane 2 : Colon (Rat) Tissue Lysate
Lane 3 : HCT 116 (Human Colorectal Carcinoma) Whole Cell Lysate
Lane 4 : SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate
Lane 5 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
Lane 6 : HuES7 (Human embryonic stem cell line) Whole Cell Lysate
Lane 7 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : 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
Additional bands at: 40 kDa. We are unsure as to the identity of these extra bands.

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

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

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