Anti-Choline Acetyltransferase抗体(ab6168)

Thank you for your reply.

We have not validated ab6168 for use on perfused-fixed frozen sections (IHC-FoFr), and it would not be guaranteed for use in that application. The alternative antibody ab18736 is known to work well on perfused-fixed rat sections and may be a better choice for this particular application. In the future, if you are interested in testing one of our products in an untested species or application, please contact us in advance and we will be happy to check whether it will be eligible for our 100% testing discount program:

https://www.abcam.com/index.html?pageconfig=resource&rid=11998

For the current experiments, I would recommend trying a shorter fixation such as < 30 minutes in PFA or using ice cold methanol or acetone, without the perfusion fixation. This will avoid the extensive cross-linking caused by perfusion fixation and would be covered under our guarantee for IHC-Fr. If you cannot change the fixation and must perfusion-fix your samples, antigen retrieval may be required. I would recommend trying the following protocol for IHC-FoFr:

IHC-FoFr Protocol:

Permeabilize thicker sections using 0.1% Triton X-100 in TBS for 10 minutes.
Perform antigen retrieval using enzymatic or heatmediated methods (see the attached reference for details on performing heat mediated antigen retrieval on frozen tissue).
Block sections with 10% normal serum from the host species of your secondary antibody in TBST (TBS + 0.1% Tween 20) for 30 minutes. If using HRP / DAB detection, also perform an endogenous peroxidase block with 3% H2O2 in 30% methanol for 10 minutes.
Incubate the sections with primary antibody ab6168 at a dilution of 1:50-1:100 overnight at 4C. Wash 3 x 5 minutes in TBST.
Incubate the sections with an anti-rabbit IgG secondary antibody according to the manufacturer instructions (˜1:500forHRPconjugatedsecondaries) for 1 hour at room temperature.
Apply enzymesubstrate according to themanufacturer's instructions. Mount with a media appropriate for yourmethod of detection, and coverslip.



If you observea weak signal, it mayhelp to usefluorometricdetection oran ABC amplification kit.This protocol is a recommendation, but as I mentioned, we have not validated ab6168 for use in IHC-FoFr and it may not be suitable for that application. Please let me know if you have any further questions or concerns.

Thank you for your email. I am sorry that this antibody is giving a weak signal. Could you please clarify a few points of your protocol?

1) What type of samples were tested? Were these rat brain sections or some other type of tissue? How long were the samples fixed in 4% PFA? Were the rats perfusion fixed or just post-fixed?

2) For the antigen retrieval, what temperature was used and how long were the slides incubated? In general, when weak signal is observed it often helps to increase the retrieval time. When high background is observed, the retrieval time should be reduced.

3)How many wash steps were performed after the primary and secondary antibody incubations? Washing for too long can often reduce the signal. I typically recommend washing three times for five minutes each.

4)Could you please clarify the secondary antibody dilution? The document you sent listed "Secondary antibody 250ul, 10%Triton-100 30ul, 100%NGS 10ul,PBS 710ul," which would indicate a 1:4 dilution. Was the antibody further diluted to 1:250 - 1:1000, or was this stock solution further diluted? Were you using a goat anti-rabbit IgG secondary antibody?

I hope this helps, if not, please let me know and I will be happy to assist you further .

Thank you for contacting Abcam. I am sorry that you are having problems with ab6168 in IHC-P. I found the following reference that used the antibody successfully in IHC-P on mouse tissue: Tu JL et al. APOE 4 polymorphism results in early cognitive deficits in an EAE model. Biochem Biophys Res Commun 384:466-70 (2009). http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=19422789&dopt=Abstract The protocol that they used in the paper is as follows: After the water maze test, mice were euthanized by terminal anesthesia for immunohistochemistry. They were perfused by ice-cold 4% paraformaldehyde fixative. Upon completion of perfusion, brains were removed and fixed in methacarn (methanol 6: chloroform 3: acetic acid 1) solution for 24 h and then embedded in paraffin. Paraffin-embedded sections were cut at 6 μm and mounted on Silane-coated standard glass microscope slides http://www.sciencedirect.com/science/article/pii/S0006291X09008717#ref_bib12. Standard H&E staining were performed before immunocytochemistry for gross morphological assessment. To identify cholinergic neurons, brain sections were incubated with primary antibody (anti-ChAT Abcam Inc., Cambridge, MA) at optimized concentration of 1:2000 at 4 °C for 16 h. Immunolabeling was detected by applying the peroxidase–antiperoxidase procedure with 3,3′-diaminobenzidine as cosubstrate http://www.sciencedirect.com/science/article/pii/S0006291X09008717#ref_bib13. I know that the protocol information they give is not complete but I was wondering if it is similar to the protocol that you are using. If not, would you be able to send me a copy of the protocol that you are using and I will see if there is any changes I can suggest. The antibody is covered under our Abpromise for six months and is guaranteed to work in IHC-P on mouse tissue. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund. I look forward to your response and helping you resolve this issue.

Thank you very much for your interest in our products. The following antibodies are tested and guaranteed for IHC-P in rat. Unfortunately, we do not have blocking peptide for these antibodies in our catalogue. ab68779 Click here (or use the following: https://www.abcam.com/Choline-Acetyltransferase-antibody-ab68779.html). ab6168 Click here (or use the following: https://www.abcam.com/Choline-Acetyltransferase-antibody-ab6168.html). ab70219 Click here (or use the following: https://www.abcam.com/index.html?datasheet =70219). ab18736 Click here (or use the following: https://www.abcam.com/Choline-Acetyltransferase-antibody-ab18736.html). ab34419 Click here (or use the following: https://www.abcam.com/Choline-Acetyltransferase-antibody-ab34419.html). The following antibodies have a blocking peptide readily available, but are not tested and guaranteed for IHC-P. Therefore you might be eligible for our testing discount program if you would like to test one of these antibodies in IHC-P on rat samples. ab85609 with ab97420 Click here (or use the following: https://www.abcam.com/Choline-Acetyltransferase-antibody-C-terminal-ab85609.html). ab50412 with ab50440 Click here (or use the following: https://www.abcam.com/Choline-Acetyltransferase-antibody-ab50412.html). ab27484 with ab45678 Click here (or use the following: https://www.abcam.com/Choline-Acetyltransferase-antibody-ab27484.html). Please do not hesitate to contact me again with any further questions or if you consider testing one of the latter antibodies in IHC-P.