ab10297 at 1/100 staining U20S cells. The cells were paraformaldehyde fixed and blocked with BSA prior to incubation with the antibody for 30 minutes. A FITC conjugated anti-mouse antibody was used as the secondary. The cells were counterstained with DAPI (left hand panel).See Abreview

Anti-hnRNP U antibody [3G6] (ab10297) at 1/5000 dilution + Human 293T - whole cell lysate at 100 µgSecondaryHRP conjugated sheep anti-mouse IgGdeveloped using the ECL techniquePerformed under reducing conditions.

ab10297 (4µg/ml)) staining hnRNPU in human cerebellum using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining of Purkinje cells neurons.Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Overlay histogram showing HeLa cells stained with ab10297 (red line). The cells were fixed with 100% 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 (ab10297, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.