Anti-NeuroD1 antibody (ab16508)

Thanks again for your help. I would like to try out the NeuroD1 antibody, ab60704, if possible.

I am attaching the order receipt for the previously ordered ab16508 to this email. Please do let me know if you need more information from me.

Thank you.

ANSWER:

Thank you for providing that information.

I have sent you ab60704 as a replacement for ab16508 which did not work as stated on our datasheet. Your new order number is *********.

The new antibody is still covered under our Abpromise and so if you have any problems with it, please let us knows.

Thank you very much for your email and for your offer to send me another NeuroD1 antibody. However, before I do that, I just had a few questions. I need to use the NeuroD1 antibody to stain granule neurons in postnatal mice cerebella. However, the previous NeuroD1 antibody I used (ab16508) ended up staining many other neurons in the wild-type cerebellum (such as Purkinje neurons), and even some Bergmann glia, where the NeuroD1 protein is not known to be expressed. Furthermore, I saw little to no staining in granule neurons, where the expression and functional role of NeuroD1 protein is widely reported (image attached). This is despite trying different ages and fixation and antigen retrieval methods, as outlined in the protocols sent to me by Abcam.

Hence, I was wondering if you could recommend another NeuroD1 antibody that you know would be more specific and reliable? I would need an antibody that can be used for immunofluoresence experiments on fixed, paraffin embedded, tissue sections. Frozen sections don't work well for the mutant mouse model I'm using since I study fine cerebellar architecture that sometimes gets damaged upon cryosectioning.

I will email you detailed order information for ab16508 as soon as I get in touch with our lab technician. Thanks very much for your help! I look forward to your reply.

ANSWER:

Thank you for your reply.

I would recommend that you try ab60704:

http://www.abcam.com/NeuroD1-antibody-ab60704.html



This antibody has a lot of Abreviews and references associated with it and so you will be able to see that staining that others are getting before receiving this antibody.

If you do wish to receive ab60704, please let me know as soon as you get that information from you lab technician.

I look forward to your reply.

I ordered Rabbit polyclonal NeuroD1 antibody (ab16508) last month for my
experiments. Unfortunately, the antibody hasn't worked for me during
immunofloresence experiments on paraffin embedded sections. I have
attached a sample image I took of a sagittal section through a wild-type
P13 mouse cerebellum embedded in paraffin and stained with NeuroD1
antibody (1:500). The granule cell layer that should have been marked by
this antibody (as cited in Klisch et al. 2011) has not stained at all.
I would really appreciate if you could offer me some guidance on this.
Please do let me know if you need more information. Thank you very much
for your cooperation.

ANSWER:

Thank you for completing the follow up survey.

I am sorry that you were unsatisfied with how your complaint was handled; we do try our best to resolve any problems to the best of our ability. As you did not have any success with ab16508, I would be happy to send you another NeuroD1 antibody to try.

If you would like to test out another antibody, please let me know which one and could you also provide the original order information for ab16508.

I look forward to your reply.

Hi Abcam,I ordered Rabbit polyclonal NeuroD1 antibody (ab16508) last month for myexperiments. Unfortunately, the antibody hasn't worked for me duringimmunofloresence experiments on paraffin embedded sections. I haveattached a sample image I took of a sagittal section through a wild-typeP13 mouse cerebellum embedded in paraffin and stained with NeuroD1antibody (1:500). The granule cell layer that should have been marked bythis antibody (as cited in Klisch et al. 2011) has not stained at all.I would really appreciate if you could offer me some guidance on this.Please do let me know if you need more information. Thank you very muchfor your cooperation

ANSWER:

Thank you for contacting Abcam. I am sorry that you are having problems with ab16508. I was wondering if you had tried antigen retrieval techniques when using this antibody. I have looked through the reference you mentioned to find their protocol, which is below: "Brains were collected and fixed overnight in 4% paraformaldehyde in PBS. After cryoprotection 488 or Alexafluor 555 (Invitrogen)." They used a 1/250 dilution of ab16508 and they were not using paraffin embedded sections. Although they do not mention that they used antigen retrieval methods, I would strongly suggest using this method, as it can work really well for some antibodies. I have attached the Abcam protocol for IHC, which contains information on antigen retrieval. Please let me know if there is anything else I can help you with.

BATCH NUMBER 123803 ORDER NUMBER 157412

DESCRIPTION OF THE PROBLEM Non-specific staining. High background is also present.

SAMPLE Mouse nasal cavity tissue.

PRIMARY ANTIBODY Abcam Rabbit polyclonal NeuroD1 (ab16508) diluted in 2% BSA, 0.4% Tx-100 in PBS overnight at 4%C. Washed 3x5 minutes in 0.02% Tx-100 in PBS.

DETECTION METHOD FITC and peroxidase were used.

POSITIVE AND NEGATIVE CONTROLS USED No primary antibody sections were negative controls (very clean). Mouse nasal cavity tissue was positive control.

ANTIBODY STORAGE CONDITIONS Reconstituted in 200ul ultrafiltered dH2O. Aliquoted (10ul) and stored at -20C.

FIXATION OF SAMPLE 3% Paraformaldehyde

ANTIGEN RETRIEVAL None.

PERMEABILIZATION STEP 30 minutes in 2% BSA, 0.4% Tx-100, in PBS. Also, 3 washes in 0.4% Tx-100 in PBS following primary incubation. Secondary antibody in 0.02% Tx-100 in PBS solution.

BLOCKING CONDITIONS See above.

SECONDARY ANTIBODY Jackson Immunoresearch donkey anti-rabbit FITC (1:100) in 0.02% Tx-100 in PBS for 1 hr at room tempurature. Also tried Vectastain Elite rabbit peroxidase kit, diluting ab in 0.02% Tx-100 in PBS. Wash in PBS, dH2O.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Adding Tx-100 to wash and secondary solutions. Changing secondary/detection methods.

ADDITIONAL NOTES Will try an additional stain with mouse lung, increasing Tx-100 concentration in secondary solution.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. This is a good selling antibody that we have received some great feedback realting to its behaviour by IHC. I have read your technical questionaire and you are largely performing an approach that I would recommend. You are employing Tx-100 as a permeabilization agent; something that I am not familiar with. However, following some literature searching I can see that this agent is standard for sample permeabilization. I would appreciate it if you could provide me with details of the dilutions that you have been performing as this was not included in your protocol.

You are performing suitable washes and the only change I would like to suggest is that you reduce the dilution of the antibody in order to improve the specificity of the antibody. Please get back in touch with me should your mouse lung samples give equal high background.