ab22759 staining Fatty Acid Synthase in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab22759 at 5 µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Fatty Acid Synthase knockout HAP1 cell lysate (20 µg)
Lane 3: A549 cell lysate (20 µg)
Lane 4: Hu liver tissue lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab22759 observed at 250 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab22759 was shown to specifically react with Fatty Acid Synthase in wild-type HAP1 cells. No band was observed when Fatty Acid Synthase knockout samples were examined. Wild-type and Fatty Acid Synthase knockout samples were subjected to SDS-PAGE. ab22759 and ab18058 (loading control to Vinculin) were diluted at 1 µg/ml and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent staining for Fatty Acid Synthase in the rat striatum using Rabbit polyclonal to Fatty Acid Synthase (ab22759). Abundant staining was observed in the Striatum with lower levels of staining observed in the Corpus callosum. This is a montage of three pictures aquired using a X10 objective. ab22759 was used at 1/200 (2µg/ml) incubated overnight at room temperature.

Secondary antibody used was anti-rabbit Alexa Fluor^® 488 at 1/1000 incubated for 2 hours at room temperature. Rat brain tissue was perfusion fixed with 4% PFA followed by overnight post-fixation in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. 30µm coronal sections were cut on a cyrostat and immunohistochemistry performed by the 'free floating' technique.