Anti-Doublecortin antibody (ab18723)

Rabbit polyclonal Doublecortin antibody. Validated in WB, IHC, ICC, ICC/IF and tested in Mouse, Rat, Chicken, Cat, Human, Cynomolgus monkey, Quail, Rhesus monkey. Cited in 177 publication(s).

Overview

  • Product name
    Anti-Doublecortin antibody
    See all Doublecortin primary antibodies
  • Description
    Rabbit polyclonal to Doublecortin
  • Host species
    Rabbit
  • Specificity

    Please note: Low dilutions of this antibody can cause high background in IHC. Please use as high a dilution as possible. Optimal working dilutions are batch dependent.

  • Tested applications
    Suitable for: WB, ICC, ICC/IF, IHC-FrFl, IHC-FoFr, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cat, Human, Cynomolgus monkey, Quail, Rhesus monkey
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 300 to the C-terminus of Human Doublecortin.

    Read Abcam's proprietary immunogen policy (Peptide available as ab19804.)

  • Positive control
    • This antibody gave a positive signal in Brain (Mouse) Tissue Lysate - normal tissue, 0 days old ICC-IF: SHSY5Y cells. IHC-P: FFPE Rat brain 6 weeks/FFPE Mouse Brain 8 weeks.
  • General notes

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077).

    See other anti-rabbit secondary antibodies that can be used with this antibody.

Properties

Applications

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.

Application Abreviews Notes
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.

We recommend Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody.

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-FoFr 1/100 - 1/2000.
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.

Target

  • Function
    Seems to be required for initial steps of neuronal dispersion and cortex lamination during cerebral cortex development. May act by competing with the putative neuronal protein kinase DCAMKL1 in binding to a target protein. May in that way participate in a signaling pathway that is crucial for neuronal interaction before and during migration, possibly as part of a calcium ion-dependent signal transduction pathway. May be part with LIS-1 of an overlapping, but distinct, signaling pathways that promote neuronal migration.
  • Tissue specificity
    Highly expressed in neuronal cells of fetal brain (in the majority of cells of the cortical plate, intermediate zone and ventricular zone), but not expressed in other fetal tissues. In the adult, highly expressed in the brain frontal lobe, but very low expression in other regions of brain, and not detected in heart, placenta, lung, liver, skeletal muscles, kidney and pancreas.
  • Involvement in disease
    Defects in DCX are the cause of lissencephaly X-linked type 1 (LISX1) [MIM:300067]; also called X-LIS or LIS. LISX1 is a classic lissencephaly characterized by mental retardation and seizures that are more severe in male patients. Affected boys show an abnormally thick cortex with absent or severely reduced gyri. Clinical manifestations include feeding problems, abnormal muscular tone, seizures and severe to profound psychomotor retardation. Female patients display a less severe phenotype referred to as 'doublecortex'.
    Defects in DCX are the cause of subcortical band heterotopia X-linked (SBHX) [MIM:300067]; also known as double cortex or subcortical laminar heterotopia (SCLH). SBHX is a mild brain malformation of the lissencephaly spectrum. It is characterized by bilateral and symmetric plates or bands of gray matter found in the central white matter between the cortex and cerebral ventricles, cerebral convolutions usually appearing normal.
    Note=A chromosomal aberration involving DCX is found in lissencephaly. Translocation t(X;2)(q22.3;p25.1).
  • Sequence similarities
    Contains 2 doublecortin domains.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Database links
  • Alternative names
    • DBCN antibody
    • Dbct antibody
    • DC antibody
    • DCX antibody
    • DCX_HUMAN antibody
    • Doublecortex antibody
    • Doublin antibody
    • FLJ51296 antibody
    • Lis X antibody
    • Lis-X antibody
    • Lissencephalin X antibody
    • Lissencephalin-X antibody
    • Lissencephaly X linked antibody
    • Lissencephaly X linked doublecortin antibody
    • LISX antibody
    • Neuronal migration protein doublecortin antibody
    • OTTHUMP00000023859 antibody
    • OTTHUMP00000023860 antibody
    • OTTHUMP00000216315 antibody
    • OTTHUMP00000216316 antibody
    • SCLH antibody
    • XLIS antibody
    see all

Images

  • IHC image of ab18723 staining in Mouse 8 weeks brain formalin fixed paraffin embedded tissue section, performed on a Leica BONDTM 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.

  • 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.

    See Abreview

  • 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.

    See Abreview

  • 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 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 at 1/200 staining mouse E18 body T/S spinal cord tissue sections by IHC-P. The tissue was formaldehyde fixed and a heat mediated antigen retireval step was perfomed before incubation with the antibody for 24 hours. A biotinylated goat antibody was used as the secondary.

    See Abreview

  • 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

  • Doublecortin expression in the dentate gyrus of a 1 month-old mouse brain. Doublecortin staining using ab18723 (1/500) in the dentate gyrus of a 1 month-old mouse brain. The mouse has been perfused with paraformaldehyde 4% (50ml). After dissection, the brain has been incubated overnight in sucrose 20%, embedded in OCT and cryosectioned (10 µm). No antigen retrieval was used. The secondary antibody used was a non-Abcam Goat anti-rabbit Alexa488.
  • 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.

  • Immunohistochemistical staining (on formaldehyde/PFA-fixed paraffin-embedded sections) of Doublecortin antibody - Neuronal Marker (ab18723) on Quail Tissue sections (E6/7 brain (Saggital section). Antigen retrieval step: Heat mediated. Blocking step: 1% BSA for 10 mins at RT. Primary Antibody ab18723 incubated at 1/400 for 2 hours RT. Secondary Antibody: Biotin conjugated goat anti rabbit Ig (1/300).
  • 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.

    See Abreview

  • ab18723 staining Doublecortin in mouse dentate gyrus hippocampal tissue section by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were incubated with primary antibody 1/1000 for 16 hours at 4°C.  An Alexa Fluor®488 conjugated goat polyclonal to rabbit IgG at 1/400 dilution was used as secondary.

    See Abreview

  • Anti-Doublecortin antibody (ab18723) at 1 µg/ml + Mouse brain tissue lysate - total protein (0 days) (ab7188) at 10 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 40-45 kDa
    Observed band size: 45 kDa
    why is the actual band size different from the predicted?

References

This product has been referenced in:
  • Payne SL  et al. Initial cell maturity changes following transplantation in a hyaluronan-based hydrogel and impacts therapeutic success in the stroke-injured rodent brain. Biomaterials 192:309-322 (2019). Read more (PubMed: 30468998) »
  • Nkomozepi P  et al. Age-related changes in Ki-67 and DCX expression in the BALB/ c mouse (Mus Musculus) brain. Int J Dev Neurosci 72:36-47 (2019). Read more (PubMed: 30472241) »
See all 178 Publications for this product

Customer reviews and Q&As

1-10 of 16 Q&A

Answer

ab77450 is raised using the same immunogen as ab18723 and therefore the products are essentially identical. The only difference between these products is that batches sold under ab77450 have passed QC testing in Western Blot and ICC/IF however, in our hands, this product did not work in IHC-P. ab18723 on the other hand has been used successfully in IHC-P.

I have compared the immunogen of these antibodies with human DCLK1 and DCLK2 and found 31% homology therefore due to this low homology, we would not expect these antibodies to cross-react with DCLK1 and DCLK2.

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Answer

Thank you for contacting us. We generally recommend aliquoting our antibodies without diluting them unless indicated otherwise on the datasheet, and storing them in aliquots of no less than 10 ul. Please let me know if I can be of further assistance.

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Question
Answer

Thank you very much for your call today and for letting us know about the trouble with this antibody in IHC.

As we discussed, I'm sending a free of charge vial of ab77450 which should arrive shortly.

Please keep me updated about the results with this antibody, and let me know if there is anything else that we can do for you. Have a nice day!

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Question
Answer

Thank you for contacting us and reporting the problem with the Anti-Doublecortin antibody (ab18723).

I am sorry that the vial you received did not contain the full amount of the product and I apologise for the inconvenience this has caused. As we discussed over the phone, I have issued a free of charge replacement vial with the order number of xxxxx (purchase order number xxxxx). This should be with you by Thursday. If you have any problems receiving this please do let me know.

Please do not hesitate to contact us if you need anything further.

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Answer

Thank you very much for that information. It has been very useful in understanding the problems currently encountered with the anti-Doublecortin antibody (ab18723).
There will always be some lot to lot variability between different productions of any rabbit polyclonal antibody and some optimisation between lots can be expected. For example, the lot number GR35235-1 is of a concentration of 0.5 mg/mL whilst the lot number GR68838-1 is of a concentration of 1 mg/mL. Using the same concentrations for both lots would therefore not be appropriate.
However, I can understand your concerns and that you have tried to optimise the lot number GR35235-1. It may unfortunately be that you have received a faulty vial. As you have requested, I am able to send you a replacement vial of the lot number GR68838-1.
Would you prefer for this to be sent with the purchase order I12/008690 currently associated with the order or would you prefer for to remove this reference as it is being sent free of charge and will therefore not have an invoice associated with it?
I look forward to receiving your reply and how you would like to proceed.

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Answer

Thank you for contacting us and for alerting us to the problem you have been experiencing with one of the vials of the anti-Doublecortin antibody (ab18723) you have been using. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.
As mentioned in your email, it would be very helpful for me if you could share the images of the staining observed with the different lots used of this antibody and if possible, if you could provide a few protocol details by filling in the questionnaire attached to this email. Could you also please confirm the order number, or the approximate date of delivery?
Once I have received this information I will have look at the protocol and see if there are any suggestions we can make that may improve the results currently observed. This information will also allow us to investigate this case internally and initiate additional testing where necessary.
If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.
As requested, I have put the order xxxx on hold for the time being. I noticed there was also another order for this antibody placed the day before this one (Order number xxxxx, purchase order number xxxxx), did you want this one to go ahead as normal?
I look forward to receiving your reply.

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Question

I received an anti-DCX antibody (ab135349 a few weeks ago. I have tried a variety of of fixes and tissues, but the antibody does not work. Detailed below are the various techniques I've tried:

1. Multiple rounds of fresh-frozen cryofixed E14 brains, with sections fixed in 4% PFA or FAA for 10-20 minutes. Antigen-retrieved with and without boiling Sodium Citrate (pH 6) for 10-20 minutes. 1 hour blocking in 5% secondary-specific blocking solution with Triton, 1 hour room temperature or overnight at 4 degrees, washed 3x 5 min with PBS, 1 hour secondary (multiple ones), washed 3x 5 min PBS and coverslipped.

2. Multiple PFA-fixed E14-16 brains (heads overnight in PFA), cryofixed, cut, and stained according to above protocol.

3. FAA fixed paraffinized E16 and P30 brains, deparaffinized and antigen-retrieved with boiling sodium citrate. Staining protocol followed as above.

I've attached a few pictures of my most recent staining attempt (fresh-frozen, PFA and FAA fixed) along with staining done with the exact same secondary for a different primary antibody (same experiment, secondary control). I've also attached a picture of previous staining with your Rabbit DCX antibody at both E14 cortex and P60 hippocampus.

Could I please exchange my non-working ms anti-DCX for a rb anti-DCX (catalog #ab18723)? I should have known better than to order a different antibody than the one I know works. I've gotten great staining with your rabbit anti-DCX and would just rather have that one than try to QC another ms anti-DCX.

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Answer

Thank you for taking time to contact us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

I apologize the the attachments didn't go through. Was the trouble that you weren't seeing any staining (or high background, etc.)? Could you try sending the images againas a .doc, .ppt, .pdf, or .jpg format?

What dilution of the antibody had you tried?

I would be happy to send youa replacement of the rabbit polyclonal, ab18723, but would you be able to confirm the above? I just want to make sure we have as much information about this for our records.

I apologize for the inconvenienceand I appreciateyour cooperation. I look forward to hearing from you.

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Answer

Thank you for kindly confirming these details. I am sorry this vial of antibody has not worked for this customer.

As requested, we are pleased to arrange a free of charge replacement. I can confirm that 1 vial of ab18723 has been added to your order number #####. This has Abcam order reference #####.

I would like to reassure you that this free of charge replacement vial is also covered by our Abpromise guarantee. Should the customer still be experiencing difficulties with the new vial, or if you have any further questions, please do not hesitate to let me know.

Thank you for your help and cooperation with this case. Please do not hesitate to contact me if you need anything further.

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Question

Inquiry: 1)Antibody storage conditions (temperature/reconstitution etc) -20 °C 2)Description of the problem (high background, low signal, non-specific satining etc.): Non-specific staining and no specific signal 3)Sample (Species/Tissue/Cell Type/Cell Line etc.): Rat Brain (hippocampus) 4)Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.): Paraformladheyde perfusion followed by immersion and sucrose embedding. Frozen 40 micron sections 5)Antigen retrieval (Enzymatic method, Heat mediated technique etc.): No 6)Permeabilization step: Standard with 0.1% Triton in PBS 7)Blocking conditions (Buffer/time period, Blocking agent etc.): 2.5% normal goat serum in PBS-Triton 8)Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): ab 18723, diluted 1:1000 in blocking buffer, overnight incubation in the cold room. 9)Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step): Anti-rabbit HRP conjugared Santa Cruz or anti-rabbit conjugated with Alexa fluor 568, Invitrogen for immunofluorescence, 2 hour incubation ar room temperature. 10)Detection method: DAB for HRP conjugated secondary antibody 11)Positive and negative controls used (please specify): Positive control, differentiating neuroblasts in juvenile hippocampus. Negative control differentiated neuron in adult hippocampus. 12) How many times have you tried the IHC?: Many times with different lots of the same antibody in the past with good results and 4 times with lot GR 55971-1 with bad results. 13)Have you run a "No Primary" control?: No 14)Do you obtain the same results every time?: Yes 15)What steps have you altered? None Additional Notes We have used for more than one year different lots of the same antibody (no bottle conserved) with satisfactory rwsults (see figures marked as old ab). Storage conditions in house, protocol and material used have been always the same.

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Answer

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement with a different lot of ab18723. Or if you would prefer a credit note. If you would like for me to organise this could you please confirm the order number (or purchase order number) of ab18723.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Answer

I have been talking to the lab about ab18723 and they have told me that theres been no change in purification with this ab but it is a tricky in terms of characterising and looking at the notes, it seems there is some variability from AP to AP regarding background staining. We don't batch test this in house in FrFl IHC but we do batch test this in FFPE IHC. The 2 AP's GR55971-1 and GR52629-1 were ones that passed in the hands of a collaborator. For lot GR55971-1, it was used at a 1/1250 dilution and their notes state that a lot of tweaking was necessary to obtain a good signal in relation to background. Lot GR52629-1 was used at 1/750 dilution but also showed a little background. I think generally it's just a case of lot to variability and both these lots were passed on the basis that the specific staining is more intense than the background. I hope that helps but if you need anything else then just let me know.

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1-10 of 16 Q&A

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