Alexa Fluor® 647 Anti-Estrogen Receptor alpha antibody [SP1]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Alexa Fluor® 647 Anti-Estrogen Receptor alpha antibody [SP1] (AB267512)

Flow cytometry overlay histogram showing MCF7 cells stained with ab267512 (red line). The cells were fixed with 4 % formaldehyde (10 min) and then permeabilized with 0.1 % PBS-Triton X-100 for 15 min. The cells were incubated in 1x PBS containing 10 % normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab267512) (1x 10⁶ in 100 μl at 0.2 μg/ml (1/2500)) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Alexa Fluor^® 647 (ab199093) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 40 mW Red laser (640nm) and 670/14 bandpass filter.

This antibody gave a positive signal in MCF7 cells fixed with 80 % methanol (5 min) / permeabilized with 0.1 % PBS-Triton X-100 for 15 min used under the same conditions.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Alexa Fluor® 647 Anti-Estrogen Receptor alpha antibody [SP1] (AB267512)

IHC image of Estrogen Receptor alpha staining in a section of formalin-fixed paraffin-embedded normal human breast*.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Biocare Medical NxGen pressure cooker using retrieval settings of 110°C for 20 minutes. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature. The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab267512 at 1/100 dilution (shown in red) and counterstained using ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

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