IHC image of NCX1 staining in a section of frozen normal human kidney*. The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M 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 ab2869 at 1µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) at 1/1000. The section was then incubated with ab150117 (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488), 1/1000)) (shown in green) and ab150080 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594), 1/1000) (shown in red) for 1 hour at room temperature. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. The secondary-only control insert image is taken from an identical assay without primary antibody. The section was then mounted using Fluoromount®. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

IHC image of NCX1 staining in a section of formalin-fixed paraffin-embedded normal human kidney* performed on a Leica BOND^TM 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 20mins. The section was then incubated with ab2869, 5ug/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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

Overlay histogram showing HEK293 cells stained with ab2869 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2869, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgM (mu chain) (ab97007) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgM [ICIGM] (ab91545, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

ab2869 staining NCX1 in Human kidney tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde, permeabilized with 0.05% Tween20 and blocked with 5% normal goat serum in 1XPBS + 0.05% Tween20 for 1 hour at 25°C; antigen retrieval was by heat mediation in sodium citrate (pH 6.0) buffer. Samples were incubated with primary antibody (1/100 in blocking buffer) for 1 hour at 25°C. Ab47827 (1/500) was used as the secondary antibody.

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