Anti-Doublecortin antibody (ab18723)

IHC image of Doublecortin staining in a section of frozen PFA perfusion fixed 6 week old rat dentate gyrus. After dissection, the tissue was further fixed in PFA overnight, washed in PBS and then incubated overnight in 30% sucrose. It was then embedded in OCT and cryosectioned. Non-specific protein-protein interactions were blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M glycine and 1% (w/v) BSA for 1h at room temperature. The section was incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab18723 at 1/1300 dilution and then incubated with ab150080 (Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preabsorbed, (Shown in red) 1/1000) for 1 hour at room temperature. DAPI was used to stain the cell nuclei (blue). The secondary-only control insert image is taken from an identical assay without primary antibody.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

IHC image of Doublecortin staining in a section of frozen PFA perfusion fixed 8 week old mouse SVZ. After dissection, the tissue was further fixed in PFA overnight, washed in PBS and then incubated overnight in 30% sucrose. It was then embedded in OCT and cryosectioned. Non-specific protein-protein interactions were blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M glycine and 1% (w/v) BSA for 1h at room temperature. The section was incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab18723 at 1/2000 dilution and then incubated with ab150080 (Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preabsorbed, (Shown in red) 1/1000) for 1 hour at room temperature. DAPI was used to stain the cell nuclei (blue). The secondary-only control insert image is taken from an identical assay without primary antibody.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

IHC image of Doublecortin staining in a section of frozen PFA perfusion fixed 8 week old mouse dentate gyrus. After dissection, the tissue was further fixed in PFA overnight, washed in PBS and then incubated overnight in 30% sucrose. It was then embedded in OCT and cryosectioned. Non-specific protein-protein interactions were blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M glycine and 1% (w/v) BSA for 1h at room temperature. The section was incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab18723 at 1/2000 dilution and then incubated with ab150080 (Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preabsorbed, (Shown in red) 1/1000) for 1 hour at room temperature. DAPI was used to stain the cell nuclei (blue). The secondary-only control insert image is taken from an identical assay without primary antibody.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

Blocking buffer: 3% Milk

Gel type: MOPS

Exposure Time: 1 minute 30 seconds

ab18723 staining Doublecortin in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween 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 ab18723 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).

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

IHC-FoFr image of Doublecortin staining in Mouse adult dentate gyrus sections using ab18723 (1/100 dilution). 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 dilution). DAPI was used to stain the nuclei.

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IHC image of ab18723 staining in Mouse 8 weeks brain formalin fixed paraffin embedded tissue section, performed on a Leica BOND^TM system using the standard protocol F (with no post primary). The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) 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. Secondary-only control image is shown as insert.

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.

IHC image of ab18723 staining in rat 6 week brain formalin fixed paraffin embedded tissue section, 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 (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 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.

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Doublecortin antibody (ab18723; green) labeling cell extensions from rat embryos 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

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.

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