* Lanes 1 - 6:*Merged signal (red and green). Green - ab241548 observed at 26 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37 kDa.#ab241548 was shown to react with Bcl-2 in Western blot. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween) before incubation with ab241548 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4 °C at 0.5 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat Anti-Rat IgG H&L (IRDye 800CW) (ab253031) and Goat anti-Rabbit IgG H&L (IRDye 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

IHC image of Bcl-2 staining in a section of formalin-fixed paraffin-embedded normal human tonsil* 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 20mins. The section was then incubated with ab241548, 0.98 µg/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 hematoxylin 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.