IHC image of SSEA1 staining in Human Hodgkin’s Lymphoma formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6.0). The section was incubated with ab16285, 1/200 dilution, overnight at 4°C. A Goat anti-Mouse (IgM) HRP conjugated secondary antibody (ab97230 at 1/2000) was used to detect the primary and visualized using DAB as the chromogen. The section was 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

ab16285 stained in Mouse embryonic stem cells. Cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab16285 at 5µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) used at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

IHC image of SSEA1 staining in mouse normal brain formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol B. 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 ab16285, 10µg/ml, for 15 mins at room temperature. A Goat anti-Mouse biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC 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.