Overview

  • Product name
  • Description
    Rabbit polyclonal to SOX9
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, Sandwich ELISA, IHC-Pmore details
    Unsuitable for: ICC/IF or IHC-Fr
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Pig
    Predicted to work with: Cow, Dog, Common marmoset
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human SOX9.

    Read Abcam's proprietary immunogen policy (Peptide available as ab27845.)

  • Positive control
    • This antibody gave a positive signal in the following lysates: Neuroectoderm (HESCS differentiated) Day 5 and Day 10 - Whole Cell, Caco-2 Whole Cell, SW480 Whole Cell, Mouse Fetus (14 day old) Tissue

Properties

Applications

Our Abpromise guarantee covers the use of ab26414 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 - 2 µg/ml. Detects a band of approximately 60 kDa (predicted molecular weight: 56 kDa).
Sandwich ELISA Use a concentration of 0.1 µg/ml. For sandwich ELISA, use this antibody as Detection at 0.1 µg/ml with Mouse monoclonal to SOX9 (ab58191) as Capture.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
  • Application notes
    Is unsuitable for ICC/IF or IHC-Fr.
  • Target

    • Function
      Plays an important role in the normal skeletal development. May regulate the expression of other genes involved in chondrogenesis by acting as a transcription factor for these genes.
    • Involvement in disease
      Defects in SOX9 are the cause of campomelic dysplasia (CMD1) [MIM:114290]. CMD1 is a rare, often lethal, dominantly inherited, congenital osteochondrodysplasia, associated with male-to-female autosomal sex reversal in two-thirds of the affected karyotypic males. A disease of the newborn characterized by congenital bowing and angulation of long bones, unusually small scapulae, deformed pelvis and spine and a missing pair of ribs. Craniofacial defects such as cleft palate, micrognatia, flat face and hypertelorism are common. Various defects of the ear are often evident, affecting the cochlea, malleus incus, stapes and tympanum. Most patients die soon after birth due to respiratory distress which has been attributed to hypoplasia of the tracheobronchial cartilage and small thoracic cage.
    • Sequence similarities
      Contains 1 HMG box DNA-binding domain.
    • Cellular localization
      Nucleus.
    • Information by UniProt
    • Database links
    • Alternative names
      • campomelic dysplasia autosomal sex reversal antibody
      • CMD 1 antibody
      • CMD1 antibody
      • CMPD 1 antibody
      • CMPD1 antibody
      • SOX 9 antibody
      • Sox9 antibody
      • SOX9_HUMAN antibody
      • SRA 1 antibody
      • SRA1 antibody
      • SRXX2 antibody
      • SRXY10 antibody
      • SRY (sex determining region Y) box 9 (campomelic dysplasia autosomal antibody
      • SRY (sex determining region Y) box 9 antibody
      • SRY (sex determining region Y)-box 9 antibody
      • SRY (sex-determining region Y)-box 9 protein antibody
      • SRY related HMG box gene 9 antibody
      • Transcription factor SOX 9 antibody
      • Transcription factor SOX-9 antibody
      • transcription factor SOX9 antibody
      see all

    Images

    • Immunohistochemical analysis of formalin-fixed, paraffin embedded mouse spleen tissue, staining SOX9 with ab26414.

      Tissue was fixed with 10% Neutral Buffered Formalin and blocked with 1.5% serum for 45 minutes 21°C; antigen retrieval was by heat mediation in a Tris-EDTA buffer (pH 9). Samples were incubated with primary antibody (0.8 µg/ml in 0.3% Triton X-100 in PBS) for 1 hours at 21°C. An biotin-conjugated goat anti-rabbit polyclonal IgG (7 µg/ml) was used as the secondary antibody.

      A = Primary antibody
      B = No primary antibody

      See Abreview

    • All lanes : Anti-SOX9 antibody (ab26414) at 2 µg/ml

      Lane 1 : Neuroectoderm (differentiated from human embryonic stem cells) Day 5 - Whole Cell Lysate
      Lane 2 : Neuroectoderm (differentiated from human embryonic stem cells) Day 10 - Whole Cell Lysate
      Lane 3 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
      Lane 4 : SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate
      Lane 5 : Mouse Fetus (14 Day Old) Tissue Lysate

      Lysates/proteins at 10 µg per lane.

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

      Performed under reducing conditions.

      Predicted band size: 56 kDa
      Observed band size: 56 kDa
      Additional bands at: 100 kDa, 90 kDa. We are unsure as to the identity of these extra bands.



      Secondary antibody goat anti-rabbit HRP preadsorbed (IgG H&L; ab97080)

    • Standard Curve for SOX9 ; dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal to SOX9 (ab58191) at 5µg/ml and Detector Antibody Rabbit polyclonal to SOX9 (ab26414) at 0.1µg/ml.
    • IHC-P image of SOX9 (ab26414) on E11.5 mouse embryo sections. The sections were fixed in paraformaldehyde and underwent heat mediated antigen retrieval using Tris/EGTA (pH9). The sections were then blocked in 1% BSA solution for 30 mins at 20°C. In these images ab26414 can be seen staining sections showing spinal cord and developing cartilage (top row), developing cartilage in vertebrae (bottom row).

      See Abreview

    • Anti-SOX9 antibody (ab26414) at 1 µg/ml + Mouse Fetus (14 Day Old) Tissue Lysate at 10 µg

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

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 56 kDa
      Observed band size: 56 kDa


      Exposure time: 90 seconds

    References

    This product has been referenced in:
    • Gallina D & Lincoln J Dynamic Expression Profiles of Sox9 in Embryonic, Post Natal, and Adult Heart Valve Cell Populations. Anat Rec (Hoboken) 302:108-116 (2019). Read more (PubMed: 30412364) »
    • Sikura KÉ  et al. Potential Role of H-Ferritin in Mitigating Valvular Mineralization. Arterioscler Thromb Vasc Biol 39:413-431 (2019). Read more (PubMed: 30700131) »
    See all 44 Publications for this product

    Customer reviews and Q&As

    1-10 of 10 Q&A

    Question



    1) Abcam product code ab 26414
    2) Abcam order reference number or product batch number CCE4329146
    3) Description of the problem

    For my research I want to identify Sertoli cells in rat testes. By achieving this I perform double staining (immunohistochemical) for Sox9. I ordered the anti-sox9 antibody ab26414, but after a couple of attemps I still do not have a positive result, I just see background staining. I will give a brief description of the protocol I used:
    - deparaffinezed with xylene and ethanol
    - clean up with hydrogen peroxide in methanol, wash in PBS
    - perform antigen retrieval with sodium citrate 3x5min 98 degrees, cooling down, wash PBS
    - 5% goat serum
    - 1:100 primary antibody, incubate overnight 4 degrees, wash PBS
    - 1:200 secondary goat biotinylated antibody, incubate, wash PBS
    - 1:1000 ABC, incubate, wash PBS
    - 1:200 DAB
    - stained with hematoxylin
    I have changed the dilution of the primary antibody to 1:20 and 1:200 -> no result.
    Unfortunately, I have not made pictures of the testis. Last week I used this antibody on kidney and liver. Both should be positive controls and again I had no result. I did make pictures and those added as attachements.
    I also diluted the primary antibody 200 times, but the results were similar > no result.
    4) Sample preparation:
    Species testis = rat, kidney = cat
    Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: formalin fixed paraffin
    Sample preparation
    Positive control
    Negative control
    5) Fixation step
    Yes/No
    If yes: Fixative agent and concentration
    Fixation time
    Fixation temperature
    6) Antigen retrieval method microwaving 3 x 5min in sodium citrate buffer pH 6
    7) Permeabilization method:
    Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?
    Permeabilizing agent and concentration:
    8) Blocking agent (eg BSA, serum…): goat serum
    Concentration 5%
    Blocking time 30min
    Blocking temperature room temperature
    9) Endogenous peroxidases blocked? 3% hydrogen peroxide in methanol 30 min at room temperature
    Endogenous biotins blocked? 1:1000 ABC 60min at room temperature
    10) Primary antibody (If more than one was used, describe in “additional notes”) :
    Concentration or dilution Used several : 1:20, 1:100, 1:200
    Diluent buffer PBS/BSAc
    Incubation time overnight at 4 degrees
    11) Secondary antibody:
    Species: goat
    Reacts against: rabbit
    Concentration or dilution 1:200
    Diluent buffer PBS/BSAc
    Incubation time 60min at room temperature
    Fluorochrome or enzyme conjugate
    12) Washing after primary and secondary antibodies:
    Buffer PBS
    Number of washes after primary 6 times 5 min on shaker and after secondary 6 times 5 min on shaker
    13) Detection method 1:200 DAB and finally hematoxylin
    14) How many times have you run this staining? 5 times
    Do you obtain the same results every time? yes
    What steps have you altered to try and optimize the use of this antibody? Different dilutions
    Document attachment: Attaching images of your IHC is strongly recommended and can greatly speed up our investigation of your problem.
    Please send this questionnaire by e-mail by cutting and pasting the text in your e-mail (no attachments other than images) to technical@abcam.com.

    Read More
    Answer

    Thank you for your enquiry regarding ab26414 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

    Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

    1) Abcam order reference number or product batch number CCE4329146

    This is not batch neither order number. Please check your record again and give us the requested information. The batch number should be on the label of the tubes and the order number on the package you received in the box.

    I look forward to hearing from you soon.

    Read More

    Question
    Answer

    Thank you for your enquiry regarding ab26414 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

    Regarding specific markers for Sertoli cells, you may need to conduct some search on PubMed. I have found this paper which may help you:

    Differentiation markers of Sertoli cells and germ cells in fetal and early postnatal human testis.

    http://www.ncbi.nlm.nih.gov/pubmed/15597196

    If you wish me to take your complaint further, could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

    Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

    Read More

    Answer

    Thank you for contacting us yesterday in regards to the anti-SOX9 antibody (ab26414) and whether it would be likely to be suitable in IHC of frozen sections.

    As discussed over the phone, I have contacted the lab to ask where the information came from that the antibody was not suitable for use in this application.

    I have now had a response to this question and the information was from some testing which was performed in 2007 by one of our collaborators which showed no staining at 1/100 dilution with mouse cerebellum.

    As discussed over the phone, as you already have the antibody I would encourage you to try the staining to see what you find. We have only tried this experiment once and it may be that it just didn't work that time but that the antibody is suitable. We would be very interested to hear how you get on and if you would like any help in optimising the protocol please do let me know.

    I hope this information has been of some help. If you have any further questions, please do not hesitate to contact us again.

    Read More

    Question
    Answer

    Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for a new lot of anti-Sox9 ab26414 with the order number xxxx. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. As you have requested, I have also attached the paper referenced in our datasheet for ab58655 anti-SCXA. As we have not yet tested ab58655 in IHC-P, it is not currently covered under our Abpromise guarantee. However I would like to offer you enrollment in our 100% Abreview program. For UNTESTED species and/or applications, we have established this testing discount program. Here is a brief description of how it works: The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount. This code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for IHC-P and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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    Answer

    Thank you for contacting us with your question.

    Ab26414 works well with formalin-fixed, paraffin-embedded sections but it has not been sucessfully used with cryosections, so I wouldn't recommend using it in IHC-Fr.

    Ab3697 has been tested with frozen tissue sectionsaccording toa journal article (attached to this email, see page 33 for an image) but the tissue was fixed with methanol. We haven't tested ab3697 with PFA-fixed tissue, but it is possible that it will work. If the tissue is fixed with PFA for more than 15-20 minutes, then I would recommend a heat-mediated antigen retrieval step.

    I hope this information will be useful, but please let me know if you have any further questions or if there is anything else that we can do for you and I'll be happy to help.

    Read More

    Answer

    Thank you for submitting your Abreview.
    I trust that you have received notification of its publication on our website. I just wanted to follow up with you as you noted problems with the specificity of this vial of ab26414. I agree your data shows non-specific staining using ab26414. I apologize for this trouble.
    If you have received this antibody within 6 months of submitting your review I would be happy to offer a replacement, credit or refund. Please provide me with the PO number or Abcam ordere reference number associated with this purchase so I can process your request.

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    Question
    Answer

    Thank you for taking time to complete our questionnaire. I am sorry to hear that 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.

    Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab26414 Anti-SOX9 antibody. I would also appreciate if you can confirm some further details:

    1. According to the questionnaire, the slides show a non-specific staining. I am wondering if the endogenous biotin has been blocked when you are working with a biotin-avidin amplification system as testis contains endogenous biotin?

    2. Triton is quite a harsh detergent and usually only necessary when you want to permeabilize the tissue, but as a HIER was performed this should be obsolete. Triton can be quite harsh on some epitopes, therefore, you may increase the clarity of the staining by reducing the Triton concentration or skipping it altogether.


    Should the suggestions not improve the results, please do let me know.

    In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

    I hope this information is helpful, and I thank you for your cooperation.

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    Answer

    Gracias por contactarnos.

    Varios marcadores pueden usarse para la detección de condrocitos mediante immunohistoquímica. Solo algunos de ellos se han testado en especie canina, el resto me temo que no se han testado, por lo que se desconoce su funcionamiento en ellas.

    Le indico algunas posibilidades con los números de catálogo de Abcam:

    Collagen II: podría usarse ab300 o ab34712. Ninguno de ellos se ha testado en perro. Si está interesado en usarlo en esta especie, quizás le interese participar en el sistema Abreviews de evaluación de anticuerpos, por el cual les ofrecemos un anticuerpo completamente gratuito al mandarnos los resultados de testar el anticuerpo en la nueva especie. Si le interesa, por favor, comuníquemelo antes de realizar el pedido.

    Para saber más sobre el sistema Abreviews: https://www.abcam.com/abreviews

    Aggrecan: ab3778. Tampoco testado en perro, pero susceptible de evaluar su aplicación.

    CD29: ab30388. En este caso tampoco hay datos de su uso en especie canina, tendría que evaluarlo usted misma.

    CD44: ab19622 y ab119863. Ninguno de ellos se ha testado en perro, sin embargo, debido al elevado porcentaje de homología con dicha especie, es probable que reaccione. Por ello, si le interesa comprobarlo, puede igualmente usarlo y enviarnos los resultados, beneficiándose de una futura compra totalmente gratuita.

    SOX9: ab26414 y ab71762. En este caso, ambos anticuerpos se han usado y caracterizado en especie canina, por lo que están cubiertos por la garantía Abpromise.

    Espero que esta información le sea de utilidad. Si necesita que le enviemos una proforma de alguno o varios de los anticuerpos, si necesita más información de alguno de ellos, o si quiere saber más acerca del sistema Abreview, por favor, no dude en volvernos a contactar.

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    Question
    Answer

    Thank you for your enquiry.

    I can confirm that the immunogen for ab26414 SOX9 antibody has 23% aligment to the human SOX 10 protein. This is very low, and so the antibody is not likely to crossreact wtih SOX10. However, I am sorry this has not specifically been tested in the laboratory.

    I hope this information will be helpful to you. If you have any further quesitons, please do not hesitate to contact us.

    Read More

    Answer

    Thank you for contacting Abcam. I am sorry to hear of the difficulties that you have been experiencing with our product.

    I have had an opportunity to discuss background issue with othe IHC experts here at Abcam. While each tissue type may present its own challenges we haveno knowledge ofa consensus regarding an issue in cardyomyosites.

    I do hope that changing the fixation of the tissue will lead to better results. Please let me know if you have any questions.

    Read More

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