Anti-Coilin antibody [IH10] (ab87913)

ab87913 staining Coilin in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 ab87913 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Blocking buffer: 3% Milk

Gel type: MOPS

The band observed in the CRISPR-Cas9 edited lysate lane below 75 kDa is likely to represent a truncated form of COIL. This has not been investigated further and the functional properties of the gene product have not been determined.

ab87913(1/2000) staining Coilin in assynchronous HeLa cells (green). Cells were fixed in methanol (this image) or paraformaldehyde (see abreview image), permeabilized with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

See Abreview

IHC image of Coilin staining in human testis formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab87913, 3µg/ml overnight at +4°C. An HRP-conjugated secondary (ab97250, 1/500 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

The inset negative control image is taken from an identical assay without primary antibody.

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

IHC image of Coilin staining in Human normal testis formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab87913, 5µg/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.