All lanes : Anti-HSF1 antibody [10H8] (ab61382) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Hsf1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : K562 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 57 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab61382 observed at 57 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab61382 was shown to recognize in wild-type HAP1 cells as signal was lost at the expected MW in Hsf1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Hsf1 knockout samples were subjected to SDS-PAGE. Ab61382 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rat IgG H&L (IRDye® 800CW) preabsorbed and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HeLa cells stained with ab61382 (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 (ab61382, 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 IgG1 [RTK2071] (ab18412, 1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

IHC image of HSF1 staining in Human normal testis 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 ab61382, 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.