Anti-DDIT3 antibody [9C8] (ab11419)

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before ab11419 and ab181602 (Rabbit anti GAPDH) were incubated overnight at 4°C at a 5ug/ml concentration and 1/10000 dilution respectively. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

IHC image of DDIT3 staining in a section of formalin-fixed paraffin-embedded human pancreas adenocarcinoma* performed on a Leica BOND^TM system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab11419, 5 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.

The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab11419 staining DDIT3 in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells +/- Tunicamycin (1.5μM, 6 hours). 

The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Triton-X for 5 mins 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 ab11419 at 5μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

IHC image of DDIT3 staining in a section of formalin-fixed paraffin-embedded normal human testis* performed on a Leica BOND^TM system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab11419, 5 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.

The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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

Treated with 20µg/ml poly(I:C).

See Abreview

See Abreview

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.