IHC staining of ab3368 on formalin-fixed paraffin-embedded human liver tissue. Following antigen retrieval using Sodium Citrate H.I.E.R., the tissue was incubated with a 1/20 dilution of the primary antibody for 60 minutes at room temperature. After incubation with biotinylated anti-rat antibody, the tissue was labeled with BioLegend's HRP labeling reagent followed by addition of DAB chromogen for detection and hematoxylin counterstaining, according to the protocol provided. The image was captured with a 40X objective. Scale bar 50 µm

ICC/IF image of ab3368 stained HepG2 cells. The cells were 100% methanol fixed (5 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 (ab3368,5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rat 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.

IHC image of ab3368 staining in human lung 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 ab3368, 1µg/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.