Anti-Sca1 / Ly6A/E antibody [E13 161-7] - Hematopoietic Stem Cell Marker (ab51317)

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?

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.

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).

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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

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.

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.

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