Anti-HAND2 antibody - Carboxyterminal end (ab60037)

Phone call reporting problems in performing IHC-P with ab60037.

ANSWER:

Thank you for calling us and for alerting us to the problem you are experiencing with the anti-HAND2 antibody (ab60037). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As we discussed on the phone, I have attacheda questionnaireto this emailso thatI can gather further information regarding the samples tested and the protocol used. OnceI have receive the completed questionnaire,I will look at the protocol and see if there are any suggestionsI can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary.

I am also looking into exactly how staining of paraffin embedded tissue sections was performed previously.

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.

I look forward to receiving your reply.

Thank you for your reply. I have put the answers to your questions below and have attached a sample image.  Also, I have included a pdf of the protocol that was used.  Thank you for your help.

1) Is the secondary antibody compatible with the primary antibody?

Yes, the secondary antibody is a biotinylated horse anti rabbit IgG.  We then use a biotin/avidin/HRP kit for amplification and a DAB substrate.

2) Have you performed indirect staining (without amplification)?

We have performed the staining without amplification using a fluorescent secondary, which yielded a similar result.  All cells of the shark labeled positively with the dHand antibody.

3) Since the host species of the secondary antibody seems to be horse, I wonder if the non-specific binding sites have been blocked with 10% horse-serum?

I did a negative control in which sections received everything (including the secondary antibody and avidin/biotin/HRP solutions and DAB)  except the primary antibody.  These sections did not have any positive labeling.

4) Have you blocked any endogenous biotin and/or peroxidase?

Sections were incubated in 0.1% H2O2 in TBS to knock down endogenous peroxidase.  Nothing was done to block endogenous biotin because the negative control sections that did not recieve primary antibody did not exhibit background labeling.

ANSWER:

Thank you for getting back to me and for providing some further information.

To our knowledge, ab60037 has not been tested in catshark - only in human. The immunogenic sequence of ab60037 shares 93% homology with catshark and it is very surprising that ab60037 does not detect the catfish protein despite the high homology.

I understand that my colleague in the US office has offered a Discount code for you for testing this antibody in IHC.

The protocol looks absolutely fine to me and I would advise you to submit an Abreview to activate your code.

If you need any further assistance in the future, please do not hesitate to contact me.

Dear Sir or Madame,

We have recently purchased the anti-dHand antibody ab60037 from abcam and are trying to get it to work in a nonmodel organism (shark).  I know from in-situ data where dHand should be and am using an embryo of appropriate age.  In brief, I am staining 4% PFA fixed cryosections of shark.  The block I am using is 10%HS 1%BSA in TBS-triton (0.025% triton).  I am also performing an amplification with streptavidin/biotin.  The secondary antibody I am using is made in horse.  The reporter is Horse Radish Peroxidase with DAB.

I have found the following:

All cells of sections stained with ab60037 stain positively.

Varying the concentration of ab60037 (1:50, 1:200, 1:500) has resulted in different staining intensities, but no difference in staining distribution (i.e., all cells label but are much darker with 1:50 than 1:200).

Sections that receive no primary antibody as a negative control are completely clean--no positive labeling. 

Sections that receive a skeletal muscle antibody as a positive control stain normally with little background.

This indicates to me there is an issue with the blocking step for ab60037.  Please let me know if you have any suggestions for what I can do differently to help with this.  Is there any block specific to dHand that I can use?  Also, do you sell premade positive control slides for dHand?  Thank you for your attention to these questions.

Regards,

ANSWER:

Thank you for contacting Abcam Technical Team 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 our product.

This antibody has only been tested in human only and its cross-reactivity with other species has not been determined yet. However, it may well be that ab60037 recognizes the heart and neural crest derivatives expressed protein (2) in other species as well.

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. Could you complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response. I am particularly interested to know the followings:

1) Is the secondary antibody compatible with the primary antibody?

2) Have you performed indirect staining (without amplification)?

3) Since the host species of the secondary antibody seems to be horse, I wonder if the non-specific binding sites have been blocked with 10% horse-serum?

4) Have you blocked any endogenous biotin and/or peroxidase?

I look forward to hearing from you and hope to solve this problem as soon as possible.