ICC/IF image of ab90458 stained HeLa 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 (ab90458, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96887, DyLight® 488 goat anti-rat IgG (H+L) used at a 1/250 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
Western blot - GRP94 antibody [9G10] (ab90458)
All lanes : Anti-GRP94 antibody [9G10] (ab90458) at 1/1000 dilution
Lane 1 : GRP94 recombinant protein at 0.1 µg Lane 2 : HeLa cell lysate at 20 µg Lane 3 : Mouse liver lysate at 20 µg Lane 4 : Vero cell lysate at 20 µg
Overlay histogram showing HeLa cells stained with ab90458 (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 (ab90458, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.
IHC image of GRP94 staining in human pancreas formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab90458, 5µg/ml, for 15 mins at room temperature. A goat anti-rat 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.