Overview

  • Product name
    Anti-Sca1 / Ly6A/E antibody [E13 161-7]
    See all Sca1 / Ly6A/E primary antibodies
  • Description
    Rat monoclonal [E13 161-7] to Sca1 / Ly6A/E
  • Host species
    Rat
  • Specificity
    This antibody reacts with an antigen expressed on mouse hematopoietic stem cells.
  • Tested applications
    Suitable for: ICC/IFmore details
    Unsuitable for: WB
  • Species reactivity
    Reacts with: Mouse
  • Immunogen

    Tissue/ cell preparation of Mouse Sca1/ Ly6A/E.

    Read Abcam's proprietary immunogen policy

  • Positive control
    • ICC/IF: TH2 cell line.
  • General notes

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab51317 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
ICC/IF Use a concentration of 5 µg/ml.
  • Application notes
    Is unsuitable for WB.
  • Target

    • Function
      T-cell activation.
    • Tissue specificity
      Widely expressed.
    • Sequence similarities
      Contains 1 UPAR/Ly6 domain.
    • Post-translational
      modifications
      O-glycosylated. Not N-glycosylated.
      Not phosphorylated.
    • Cellular localization
      Cell membrane.
    • Information by UniProt
    • Database links
    • Alternative names
      • Ly-6A.2/Ly-6E. antibody
      • Ly-6A.2/Ly-6E.1 antibody
      • Ly6a antibody
      • LY6A_MOUSE antibody
      • Ly6al antibody
      • Lymphocyte antigen 6A-2/6E-1 antibody
      • Lymphocyte antigen Ly 6A.2/Ly 6E.1 antibody
      • SCA-1 antibody
      • Stem cell antigen 1 antibody
      • T cell activating protein antibody
      • T-cell-activating protein antibody
      • TAP antibody
      see all

    Images

    • ab51317 stained TH2 cells. The cells were 4% formaldehyde fixed for 4 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51317 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (ab150165) used at a 1/1000 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

    • Negative control cell line:

      ab51317 showing no staining in NIH-3T3 cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51317 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (ab150165) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

    References

    This product has been referenced in:
    • Schwalie PC  et al. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature 559:103-108 (2018). Read more (PubMed: 29925944) »
    • Zeng L  et al. Long non-coding RNA MALAT-1 contributes to maintenance of stem cell-like phenotypes in breast cancer cells. Oncol Lett 15:2117-2122 (2018). Read more (PubMed: 29434914) »
    See all 21 Publications for this product

    Customer reviews and Q&As

    1-9 of 9 Abreviews or Q&A

    Answer

    I agree that the staining seems incorrect. In two of the images, a subset of cells at the edges of the sections are staining, and in the third, the staining is of something other than cells, maybe extracellular matrix.

    The following article used both of these clones, D7 (ab25195) and E13 161-7 (ab51317), for staining mouse testis. (They were purchased from a different vendor).

    Biol Reprod. 2005 Oct;73(4):634-8. Epub 2005 Jun 1. PMID: 15930324
    http://www.biolreprod.org/content/73/4/634.long

    The staining in figure 1 is of a few different cell types but nothing like what you see.
    More relevant to your tissues is an article listed as a reference in the van Bragt paper.

    Proc Natl Acad Sci U S A. 1989 Jun;86(12):4634-8. PMID: 2660142
    http://www.pnas.org/content/86/12/4634.long


    It shows staining of Sca1/Ly6a in mouse heart in figure 5. It is not clear what antibody they used but the authors describe staining patterns for a variety of tissues:


    In three organs a specific staining with anti-Sca-1 antibody was found: in spleen the red pulp area showed a stronger staining than the white pulp area; in thymus the medulla but not the cortex reacted strongly; and in kidney the majority of the tubules were strongly reactive. In brain, heart, and liver the reaction of the antibody was restricted to blood vessels.


    I am not sure what to suggest for a protocol modification. Have you been able to stain these same tissues with other antibodies for other proteins using your protocol?

    Read More

    Question

    High background in IHC of frozen mouse heart tissue.

    I have tried the following concentrations: 1:50, 1:100, 1:200 and 1:500
    The tissue is fixed in 4% PFA/1xPBS for 3h due to GFP expression in the tissue.
    I have stained these tissues with GATA4, c-kit, phospho-histone H3 and actin. These antibodies work fine with the same blocking procedures. I use normal serum for goat or donkey (depending on the secondary).

    I tried blocking with 10% serum, 20% serum in 1%BSA/0.3M glycine/0.1% tween-20 in 1xPBS. I have also tried these mixtures without BSA. I also tried an antibody retrieval method but that destroyed all signal in the tissue. I also did a primary only and secondary only. I have tried the secondary at 1/1000 up to 1/3000 and that seems to be fine. I am wondering if there are any other blocking techniques that might work to lower the background.




















    https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header








    Dear Melissa








    Almost forgot: what concentration(s) of the anti- Sca1 / Ly6A/E primary antibody have you tried?






    Best regards,
    Thomas

    Thomas Ruyle
    Scientific Support Specialist
    Abcam Inc.
    www.abcam.com










    Your original inquiry to Abcam:



    I am having a little difficulty with antibody ab51317. I am using an Alexa fluor molecular probe secondary (IgG, goat anti-rat) for detection. The primary tissue I want to use for detection is frozen heart tissues from mice at days 0 to 62 days of age. I purchased this antibody about a month in a half ago. When I received it I aliquoted the antibody into tubes, 10ul each and kept at -20C.
    The problem I am having is with background. I tried blocking with 10% serum, 20% serum in 1%BSA/0.3M glycine/0.1% tween-20 in 1xPBS. I have also tried these mixtures without BSA. I also tried an antibody retrieval method but that destroyed all signal in the tissue. I also did a primary only and secondary only. I have tried the secondary at 1/1000 up to 1/3000 and that seems to be fine.
    I am wondering if there are any other blocking techniques that might work to lower the background.

    Thank you for any help you can provide,
    Melissa Jackson







    [CCE4595247]

    SID:42








    Discover more at abcam.com

    Read More
    Answer

    How do the stains with 1/500 compare to the other dilutions? Is the background intensity the same? Do you see anything that appears to be a specific stain?

    One reagent that is sometimes added to blocking buffers is glycine, to block free aldehyde groups that may react with the primary antibody. The serum proteins should do this but glycine is much smaller and the rationale for using it is that it can diffuse to areas that large proteins cannot.

    Our lab uses glycine in a blocking buffer at 0.3M concentration. As I mentioned, we have not tested this antibody on sections of frozen tissue, only paraffin-embedded. There may be something about the paraffin processing that prevents the occurence of whatever it is that is causing the background you are seeing. On the other hand, it may be that the antibody is defective. Although our guarantee is only offered for applications listed on the datasheet, for example IHC-P (IHC of paraffin-embedded tissue), I will replace this antibody for you if the glycine blocking buffer (with 5-10% serum) does not help.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (Bone marrow)
    Specification
    Bone marrow
    Fixative
    Formaldehyde
    Permeabilization
    No
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Apr 12 2012

    Answer

    Thank you for you reply.

    Your credit note IDs are ***** & *********.

    I am sorry that these antibodies did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you for all of these antibodies (ab2074, ab5506, ab32392, ab39707, ab51317, ab52971 and ab65006).

    The credit notes may be used in one of the following ways:

    (1) Redeemed against the original invoice if this hasn't already been paid.
    (2) Held on the account for use against a future order.
    (3) A full refund can be offered where no other invoices are outstanding.

    Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

    To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

    The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

    I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

    Read More

    Question

    We've purchased a number of antibodies from AbCam over the course of the past 6 months and we're struggling to get them to work properly. We therefore wonder if you could provide us with some advice to improve their performance. The antibodies in question and their respective dilutions are:
    CXCR4 - ab2074 (1:100)
    EpCam - ab32392 (1:250)
    LRIG1 - ab36707 (1:200)
    Integrin beta1 - ab52971 (1:250)
    TACD2 - ab65006 (1:200)
    c-Kit - ab5506 (1:100)
    Sca1 - ab51317 (1:100)
    Our samples are murine tumour and normal tissue from Skin, Mammary and Prostate (only Prostate for the last three antibodies). All samples are Formalin fixed and Paraffin embedded. We use a generic immunohistochemistry protocol. Antigen retrieval is performed by heating slides in Dako Citrate buffer at 100C for 20 min. TBS+Tween20 is used as washing solution. The blocking agent (for rabbit antibodies) is 10% normal goat serum. Antibody binding is detected by anti-rabbit HRP-conjugated secondary antibody from Envision kit with subsequent visualisation with DAB reagent. Rabbit anti-rat biotinylated secondary in 10% normal rabbit serum was used to detect Sca1 primary followed by ABC kit (vectastatin) and DAB treatment.

    The main problems we've encountered with the named antibodies are the lack of positive staining (for c-Kit and Sca1 antibodies) and non-specific staining (for the rest). CXCR4, LRIG1, EpCam, Integrin beta 1 and TACD2 all displayed nuclear staining of various intensity instead of expected membrane-associated staining pattern.

    We would therefore appreciate any suggestions on potential ways to improve the staining. Please, let me know if you would like me to send you detailed protocols and/or images of the stained tissue.

    Read More
    Answer

    Thank you for contacting Abcam.

    I am sorry about all of the problems you have been having with these antibodies.

    It would greatly be appreciated if you could send me some of the images that you are getting using these antibodies and also could you answer the questions below, so that I can understand your protocol a little more:

    1 - How long do you block your samples for?

    2 - How long do you incubate your primary antibodies for?

    3 - Do you use a hydrogen peroxidase step?

    4 - Are your samples permeabilised? If so with what percentage of detergent and for how long?

    5 - Have you tried testing any of these antibodies without antigen retrieval, as for ab2074 & ab36707 at least, we have some information that suggests antigen retrieval is not necessary.

    Once again, I am sorry about all the problems you have been and having and I hope to able to help you resolve this issue.

    I look forward to your reply.

    Read More

    Answer

    Thank you for contacting us. Because we carry over 80,000 products, it isn't feasible for us to keep small sample sizes of our products.

    We are happy to reassure our customers that all of our products - including the Sca1/Ly6A/E antibody ab51317 - are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet (i.e. IHC-P and ICC/IF on mouse), or we will offer a replacement, credit, or refund within 6 months of purchase.

    If the product is to be used in an untested species or application, you may be eligible for our testing discount program if the antibody has not yet been purchased (www.abcam.com/collaborationdiscount). Please contact our Scientific Support team by replying to this email prior to purchase for more information.

    Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates. To find out more about our Abreview system, please see the following link: https://www.abcam.com/abreviews

    I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

    Read More

    Answer

    Vielen Dank für diese zusätzlichen Informationen.
    Ich würde Sie bitten die folgenden drei Tipps auszuprobieren und möchte Ihnen nochmals versichern, dass wenn die Protokolltipps das Resultat nicht verbessern, die Abpromise Garantie greift und ich Ihnen alternative Antikörper oder eine Rückerstattung anbieten kann.
    1.) Wir möchten vorschlagen einen "time course" für die Antigendemaskierung zu machen. Je nach Länge der Fixierungszeit, muss die Demaskierung angepasst werden. Leider sind bei der Antigendemaskierung keinerlei Regeln bekannt und es muss ausprobiert werden.
    2.) Da es sich bei allen Ihren Zielproteinen um Membranproteine handelt, empfehlen wir kein Permeabilisierungsreagenz zu verwenden. TWEEN wäscht Lipide aus der Membran aus und das könnte die Konfirmation der Markerproteine verändern.
    3.) Wir möchten auch empfehlen, 0.2M Glycin im Blockierungspuffer zu verwenden. Damit kann der Hintergrund, den Sie in der Milz sahen, vielleicht verringert werden. Glycin legt sich auf die freien Aldehydgruppen.
    Bitte lassen Sie mich wissen, ob diese Tipps geholfen haben oder nicht. Ich wünsche Ihnen viel Erfolg und verbleibe

    Read More
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Skin)
    Antigen retrieval step
    None
    Permeabilization
    No
    Specification
    Skin
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Sep 08 2010

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Kidney)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Dako Target Retrieval Solution
    Permeabilization
    No
    Specification
    Kidney
    Blocking step
    5 minutes of peroxidase block then 10 minutes of protein block. These are ready-to-use reagents purchased from Dako as blocking agent for 15 minute(s) · Concentration: 100% · Temperature: 20°C
    Fixative
    Formaldehyde

    Mr. Antibody Solutions

    Verified customer

    Submitted Apr 08 2010

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