IHC image of Glucose Transporter GLUT4 staining in human heart formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab654, 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.

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

ab654 staining GLUT4 in human mesenchymal stem cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 10% serum for 30 minutes at 22°C. Samples were incubated with primary antibody (1/100 in 10% Horse serum) for 1 hour at 22°C. A FITC-conjugated rabbit polyclonal was used as the secondary antibody (1/100).

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ICC/IF image of ab654 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab654, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

ab654 staining GLUT4 in human endometriosis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 3% BSA for 1 hour at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/50 in 3% BSA in TBS) for 16 hours at 4°C. An undiluted Biotin-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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