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Synthetic peptide conjugated to KLH derived from within residues 300 to the C-terminus of Human Doublecortin.
(Peptide available as ab19804.)
Our Abpromise guarantee covers the use of ab18723 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 45 kDa (predicted molecular weight: 40-45 kDa).|
|ICC||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 1 - 5 µg/ml.|
|IHC-FrFl||1/1000. Please note: Low dilutions of this antibody can cause high background. Please use as high a dilution as possible. Optimal working dilutions are batch dependent.|
|IHC-Fr||1/2000 - 1/7000.|
|IHC-P||Use a concentration of 0.05 - 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
ab18723 staining Doublecortin in mouse embryonic 15 day brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde, permeablized with 0.5% Triton X-100 in PBS, blocked with 10% serum for 1 hour at 25°C and antigen retrieval was by heat mediation in citrate buffer, pH 6. The sample was incubated with primary antibody (1/500 in PBS + 0.1% Triton X-100 + 1% serum) for 16 hours at 25°C. An Alexa Fluor® 594-conjugated donkey anti-rabbit IgG polyclonal (1/700) was used as the secondary antibody.
IHC-Fr image of Doublecortin staining in Mouse adult dentate gyrus sections using ab18723(1:100). The sections were fixed in paraformaldehyde and permeabilized using 1x TBST. The sections were then blocked using 10% donkey serum for 1 hour at 25°C. ab18723 was diluted 1:100 and incubated with the sections for 12 hours at 25°C. The secondary antibody used was Donkey anti-rabbit conjugated to Cy3 Dye (1:500). DAPI was used to stain the nuclei.
ab18723 staining 6 week rat brain tissue dentate gyrus (DG) by IHC-P using rabbit-specific EXPOSE IHC detection kit (ab80437). Formalin fixed paraffin embedded tissue sections were pre-treated using heat mediated antigen retrieval (using a pressure cooker) with sodium citrate buffer (pH6) for 30 mins. The section was incubated with ab18723, 0.1µg/ml, for 1 hour at room temperature. DAB was used as the chromogen and the section was counterstained with haematoxylin and mounted with DPX.
ab18723 stained in SHSY5Y cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18723 at 5 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Doublecortin antibody (ab18723; green) labeling cell extensions consistent with dendrite morphology in 4 day old cultures. Preincubation of ab18723 with its immunising peptide (ab19804) quenched immunostaining (see review).
Dorsal root ganglion explants were dissected from 16 day-old rat embryos and cultured for 4 days in vitro with Neurobasal Medium containing 10% fetal calf serum and B27 supplement. Immunocytochemistry: All steps were performed in PBS. Cells or explants were fixed in 4% PFA for 15min, permeabilised with 0.1% TX100 for 10min and blocked with 5% BSA, 0.1% TX100 for 45min. ab18723 was incubated at 5µg/ml for 12h in 5% BSA, 0.1% TX100 at 4°C. For peptide blocking experiments preincubation of the peptide (ab19804; 250µg/ml) and antibody (5µg/ml ) was performed for 2h at 37°C. Cultures were washed (3x) of primary antibody solution. Goat anti-rabbit AlexaFluor 488 was used as secondary antibody (1/400) in 5% BSA, 0.1% TX100 for 2h at R
IHC image of ab18723 staining in rat 6 week brain formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) for 20 mins. The section was then incubated with ab18723, 0.1µg/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 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.
ab18723 at 1/2000 staining mouse brain svr: progenitor olfactory neurones by IHC-P. The tissue was formaldehyde fixed and a heat mediated antigen retrieval step was performed before incubation with the antibody for 16 hours. An Alexa-Fluor ® 488 conjugated goat antibody was used as the secondary (green). The tissue was also stained for Ki67 (shown in red).
The MIP image was derived from Apotome-generated Z-stacks from the greyscale image of each of the channels in the MIP.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"