Anti-DUSP6 antibody [EPR129Y] (ab76310)

Immunofluorescence staining of PC-12 cells with purified ab76310 at a working dilution of 1/200, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab76310 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

ab76310 detects an unspecific band around 100 kDa in human materials.

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

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Immunohistochemistry (PFA perfusion fixed frozen sections) analysis of Mouse brain tissue sections labelling DUSP6 with unpurified ab76310 at 1/400 dilution for 14 hours at 4°C. A biotinylated polyclonal anti-rabbit IgG was used as the secondary antibody at 1/250 dilution.

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Immunohistochemical staining of paraffin-embedded human gastric carcinoma using unpurified ab76310 at 1/50 dilution.

ICC/IF image of unpurified ab76310 stained PC12 cells. The cells were 4% formaldehyde fixed (10 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 (ab76310, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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.

Overlay histogram showing HeLa cells stained with unpurified ab76310 (red line). The cells were fixed with 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 (ab76310, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/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/permeabilized with 0.1% PBS-Tween 20 used under the same conditions.