Anti-Estrogen Receptor alpha antibody [SP1] (ab16660)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial cell) cells labeling Estrogen Receptor alpha with purified ab16660 at 1/25 (8.5 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

IHC image of ab16660 staining Estrogen Receptor alpha in normal human cervix formalin-fixed paraffin-embedded tissue sections*, performed on a Leica Bond. 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 ab16660, 1/250 dilution, 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. No primary antibody was used in the negative control (shown on the inset).

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

Flow cytometry analysis of MCF7 (human breast adenocarcinoma epithelial cell) labeling Estrogen Receptor alpha with purified ab16660 at 1/200 dilution (1.06 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control - Rabbit monoclonal IgG (ab172730) (black). Unlableled control - Unlabelled cells (blue).

ab16660 staining Estrogen Receptor alpha in MCF7 cells. The cells were fixed with 4% formaldehyde (10min), 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 ab16660 at 1/250 dilution (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical analysis of paraformaldehyde-fixed, 0.1% Triton 0.05% Tween permeabilized frozen Mouse Tissue sections (Intestine) stained for Estrogen Receptor alpha (purple) using ab16660 at 1/100 dilution. Primary was incubated for 16 hours at 4°C with serum as blocking agent for 2 hours at 20°C.

Secondary antibody was AffiniPure F(ab')2 Fragment Alexa Fluor^® 647-conjugated Donkey anti-rabbit IgG at 1/500 dilution.

HPRT-Cre/ERT2 x mT/mG mouse, treated with tamoxifen.

Formalin-fixed, paraffin-embedded human ovarian adenocarcinoma tissue stained for Estrogen Receptor alpha using ab16660 at 1/200 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human breast ductal carcinoma tissue stained for Estrogen Receptor alpha using ab16660 at 1/200 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human breast tissue stained for Estrogen Receptor alpha using ab16660 at 1/200 dilution in immunohistochemical analysis.

Formalin-fixed, paraffin-embedded human breast carcinoma tissue stained for Estrogen Receptor alpha using ab16660 at 1/200 dilution in immunohistochemical analysis.