Noradrenaline (conjugated) antibody (ab8887)

What is the concentration of this antibody?

ANSWER:

Thank you for contacting Abcam regarding ab8887.

I have confirmed with the laboratory that unfortunately, there is no concentration information available for this antibody.

I apologize for any inconvenience this may cause. Please do not hesitate to contact us if you have any additional questions.

On FAQ, this antibody has been used on formalin-fixed tissue sections but the reply from Abcam was that this antibody has not been tested on formalin-fixed tissue. This is in contrast to your datasheet which states that this antibody can be used on IHC-P, which according to your key is a formalin-fixed, paraffin embedded tissue.

Can this antibody be used on formalin-fixed tissue that is paraffin-embedded?

ANSWER:

Thank you for your enquiry.

The FAQ you noticed is from Thursday 25-November-2004 at which time the antibody had not been tested in IHC-P.

We since have data from a publication that came to our attention in Sept 2008. PMID 18457899. This publication has used the antibody successfully in IHC-P. We therefore added IHC-P as a tested application and I can confirm we provide our Abpromise guarantee for this antibody in IHC-P.

I hope this will be helpful to you. Should you have any further questions, please do not hesitate to contact us.

In our lab we use a phenol-ethanol supernatant obtained after precipitation of DNA with ethanol, we wonder if we could use this supernatant for the ELISA with acetilcholine and noradrenaline antibodies.

The protocols displayed in the internet page, have been usefull. I look forward to your reply.

ANSWER:

Thank you for your enquiry.

Regarding "using protein in the phenol-ethanol supernatant for ELISA", would you first precipitate the protein? In either case, I'm not sure if the protein would be recognized by the antibodies since we have not tested this ourselves.

We invite you to use our Abreview system. The Abreview system allows researchers to submit reviews of our products in exchange for Abpoints. Abpoints can be redeemed for rewards such as Amazon.com gift certificates or discounts on future antibody purchases. Should you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview right from the online datasheet.

I hope this information helps and please let me know how the ELISAs work.

Thank you for your questions. I would like to answer them one by one.

> 1. Please describe the problem (high background, no staining etc). answer: no staining, no positive results. > 2. On what material are you testing the antibody in IHC? answer: human and rat bladder Tissue > > 3. How did you fix the samples? answer: Paraformaldehyde > > 4. Did you apply antigen retrieval step? answer: I do not think this procedure is needed, since I used fresh cryostat sections. > > 5. How did you block the unspecific binding sites? answer: using normal horse serum > > 6. Primary antibody Specification (in which species was it raised against)? answer: human and rat

At what dilution(s) have you tested this antibody? answer: from 1:200, 1:400, 1:1000, 1:2000, to 1:4000.

Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? answer: incubation at 4c for 16-18 hours, or room tempereture for 16 to 18 hours. wash steps: each time 5 minutes for 3 times. > > 7. Secondary antibody What secondary antibody are you using? answer: "Histofine simple stain Max(R)" from Novocastra company or "Alexa Fluore 594 donkey anti-rabbit IgG" from Molecular Probe.

Specification (in which species was it raised against)? answer: bladder including neck and dome.

What detection method are you using? answer: both DAB staining and immunoflorencent methods. > 8. Background staining Please provide an image of your staining answer: the image is attached in the attachment. > > 9. Which detection system did you use? answer: DAB staining and immunoflorencent

> 10. Did you apply positive and negative controls along with the samples? Please specify. answer: Yes, for positive control, I used other antibodys such as TH, for staining of noradenergic nerve fibers in the same series sections, and got good positive results. For negative controls, I used the normal rabbit serum to replace the primary antibody. > > 11. Optimization attempts How many times have you tried the IHC? answer: for more than 5 times.

Do you obtain the same results every time? answer: yes.

What steps have you altered? answer: different dilution(s) of primary antibody, and different incubation times, and different decondary detecting systems.

Thank you for your comprehensive questions. I hope you will be satisfied with my answers.

I am looking forward to seeing your answer again.

ANSWER:

Thank you for your patience and forwarding your detailed protocol on-line. We are very sorry to hear that you are having some problem with this antibody.

Looking through your email, I can understand that you have fixed the tissue sections in paraformaldehyde. As a general rule, formalin fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross link proteins and therefore mask antigenic sites.

This particular antibody has not been tested for IHC on formalin-fixed sections only on frozen section. We would therefore suggest trying different fixatives: either ice-cold acetone (for 5 or 10 min), or methanol or in ethanol.

There are two specific publications listed on the datasheet, please take a look at them; you may find some useful information in them.

We need to emphasize that each antibody has different specificity and characteristics so even if another antibody (like TH you mentioned in your message) works nicely in the same series sections, it does not necessary mean that this antibody is suitable for IHC on formalin-fixed sections.

We hope this will help you. Should you still have problem with this product, please do not hesitate to contact us again.