Anti-DDIT3 antibody [9C8] (ab11419)

Immunostaining of DDIT3 (green) and AVP (red) in the supraoptic nucleus of 3 days DH and 7 days SL rats shows expression of CHOP in the nuclei of AVP MCNs (magnocellular neurons). DDIT3 was stained using ab11419 at 1/200 dilution.

The rat  brains were removed and post-fixed overnight in 4% (w/v) PFA followed by 30% (w/v) sucrose prepared in PBS to cryoprotect the tissue prior to freezing over liquid nitrogen. Coronal sections (40 μm) of the forebrain were cut on a cryostat and washed in PBS three times. Sections then were blocked in 5% fetal bovine serum prepared in 0.25% (v/v) TritonX/PBS (PBST) for 30 min and then incubated with appropriate primary antibodies at 4°C for 48 hours.

DH = Complete fluid deprivation.
SL = Salt loading by drimking 2% salt solution

Western blot analysis of HeLa cell lysates using DDIT3 monoclonal antibody (ab11419). HeLa cells were left untreated (-) or treated with tunicamycin (5μg/ml) for 11 hours (+). Membranes containing whole cell extracts were blocked with 5% milk in TBST (blocking buffer) and probed overnight at 4^oC with ab11419 (diluted 1:2000 in blocking buffer) followed by a goat anti-mouse IgG-HRP secondary antibody (diluted in blocking buffer) and a chemiluminescent substrate. Membranes were also probed for actin as a loading control.

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Overlay histogram showing HeLa cells stained with ab11419 (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 (ab11419, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b  (ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Treated with 20µg/ml poly(I:C).

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Western blot of endogenous DDIT3 from primary Human fibroblasts using ab11419.
Lane 1: Untreated cells
Lane 2: Cells treated with tunicamycin for 10 hours

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ab11419 staining DDIT3 in SKNSH cells treated with deltamethrin (ab141019), by ICC/IF. Increase of DDIT3 expression correlates with increased concentration of deltamethrin, as described in literature.
The cells were incubated at 37°C for 48 hours in media containing different concentrations of ab141019 (deltamethrin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab11419 (10 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.