Anti-GAPDH antibody [EPR6256] - Loading Control (ab128915)

Secondary antibody - anti-rabbit HRP (ab6721)

IHC image of ab128915 staining GAPDH in human pancreas* 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 ab128915, 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 secondary only 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

ab128915 staining GAPDH in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab128915 at 2μg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an goat anti-rabbit AlexaFluor®488 (ab150081) at 2 μg/ml (shown in green) and goat anti-mouse AlexaFluor®594 (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

ab128915 staining GAPDH in human HeLa cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour at 22°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody. Counter stained with DAPI.

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Overlay histogram showing HeLa cells stained with ab128915 (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 (ab128915, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was goat anti-rabbit Alexa Fluor® 488 (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.