Anti-Sca1 / Ly6A/E antibody [E13 161-7] (ab51317)

I safely received the ab5506 antibody and tried it out this weekend. I used 5% goat serum as a blocking agent this time. Unfortunately, it still produced no staining in either normal or tumour prostate. I have also tried Lrgi1 and Cxcr4 antibodies (ab2074 & ab36707) without antigen retrieval. To my surprise the staining worked, but it was still nuclear instead of predicted membrane localisation. So no improvement over the protocol with antigen retrieval. I think we'll have to go with the refund option.

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

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.

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.

Hi,I am not sure you provide samples of antibody for testing. I would be interested in this one:Mesenchymal stem cell antibody highlights Sca1 / Ly6A/E (ab51317

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: http://www.abcam.com/abreviews

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

unsere Proben werden in 5-10% gepufferter Formalinlösung über Nacht fixiert und in der Pathologie eingebettet.
Der Antikörper, der funktioniert ist anti CD 45 (rabbit) Nr. ab10558, das protokoll ist das gleiche,
weil ich alle Antikörper in einem System brauche.
Viele Grüße

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