Anti-DAZL antibody (ab34139)

I have a question regarding the rabbit polyclonal DAZL antibody (ab34139). I have been trying to get this antibody to work on mouse testis and ovary 10um sections. I do not get any signal above background (secondary alone control) I was wondering if you could tell me what your experience is.

organs were fixed in 4% paraformaldehyde overnight at 4 degrees. then in 30% sucrose overnight at 4 degrees then, embedded in OCT & frozen at -80. 10um sections were made and blocked with 3% donkey serum in PBS+0.1% triton-x at roomtemp for 30 minutes. primary DAZL antibody was added 1:100 in the described blocking solution and incubated overnight at 4 degrees. slides were washed 3x 30 minutes at roomtemperature in PBS + tween 20 before adding alexa coupled secondary antibodies. The problem is simply that I cannot get any above background control signal on either mouse testis or ovary. I see you show IHC pics on your website - is that the only way to go? thanks a lot for your help!

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you are experiencing difficulties with this product ab34139. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support but please bear in mind that this product has not been tested using frozen sections (IHC-Fr).

I have looked at the protocols you used and have a few questions and suggestions that might help you determine the cause of the no specific staining results. I would therefore appreciate if you can please clarify the following items.

Since the cellular localization of this protein is predominantly cytoplasmic (nuclear in spermatogonia until near the end of the meiotic prophase and cytoplasmic localization from then onwards), you will need to fix your samples with ice cold methanol/acetone (1-10min), which also serves to permeabilize you samples for easy detection. For protocol details, please refer to out website protocol page.

From my experience, samples prepared for frozen sections do not require overnight fixation (can be done for shorter time if desired). Usually, samples are embedded in OCT right after dissection. The reason is because usage of PFA can cause masking of the epitope and will require antigen retrieval methods. This maybe why you are not detecting any signals.

Can you confirm that the secondary alone (no-primary) control you performed, did not show any signals? Or did you get background? Did the secondary antibody also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working properly.

If the above suggestions did not help to improve your results, then I am afraid that this product is indeed not suitable for use in IHC-Fr application.

In any case, can I encourage you to submit an Abreview via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to other researchers. We reward each Abreview with Abpoints which can be used for discounts off future purchases and gifts. If you decide to submit an Abreview, please include a note that you have already spoken to me, so that I will be able to quickly publish your Abreview, as I have already tried troubleshooting with you.

To find out more about our Abreview system, please see the following URL: http://www.abcam.com/abreviews

Should you require any further information or assistance, please do not hesitate to contact me.