Anti-VIP antibody (ab8556)

Thanks so much for your reply. We will give the acetone a try then. We tried 2 different approaches thus far with the Ab on flow, only one of them has been run. It didn't look good :o/ . We used a goat anti rabbit APC secondary antibody, but perhaps it was at too high a concentration. The primary VIP antibody was used after stimulating the cells with PMA/ionomycin and brefeldin A, then permeabilizing and fixing the cells. we tried the VIP Ab at a 1:100, 200, and 400 dilution. I think we'll have to mess around more with the flow cytometry, probably just one of those things.
I will get back with you definitely about our go with the acetone!
Thank you again!

ANSWER:

Thanks for keeping me updated.

I'm sorry to hear that the flow cytometry results were not so good. We haven't tested this antibody in flow so I don't have specific recommendations for a protocol, and it might requrie some optimization as you've said. Do you know where in the cell the VIP accumulates before it is secreted? Have you considered fixing after staining with the primary antibody, or not fixing at all? Fixation might not be necessary if you're analyzing the cells immediately after staining.

I look forward to hearing from you about this and the IHC results with acetone-fixed tissue. Have a great day!

thank you so much for getting back with me. I haven't had a chance to try the auto-fluorescence methods yet, I've sort of been waiting to see what you were able to find out b/c I don't want to blow through too much of my antibody :) I will be out of town starting tomorrow, so won't be doing an experiment until next week. If you are able to uncover anything else, I would love to hear about it! If not, we will definitely try your acetone fixation suggestion, and auto-fluorescence suggestions. We were also able to get a sample of murine brain frozen hopefully as a positive tissue control.
I will definitely keep in touch with you. If nothing turns up for the IHC-frozen protocol by next week, we'll try your suggestions, and i'll let you know the results. Crossing my fingers we see something! Either way I will let you know. Today, we're also trying to get the Ab working for flow cytometry--hope it works!
Thank you so much for your help.

ANSWER:

Thanks for your reply. After further discussion with the lab, I don't think I'll find out anything else about the IHC-Fr protocol, so I would recommend going ahead and trying the acetone fixation and some of the other alterations. If you do use up the antibody while troubleshooting, I'll be happy to send another vial, so don't worry about that.

How did the flow cytometry turn out?

Please let me know if you have any questions. Have a great day!

Look forward to your response!

ANSWER:

Thanks again for your patience.

I've heard back from the lab, and they did not test ab8556 in-house on frozen tissue sections. This application was added to our datasheet based on feedback from an outside lab, and I haven't tracked down information about the IHC-Fr protocol used yet. I will remove IHC-Fr from our datasheet if I can't find more information, and I apologize for this inconvenience. My contact at the lab did recommend a fixation with ice cold acetone for 10 minutes, so you could try this fixation if you would like.

Have you had a chance to try any of the methods to reduce the auto-fluorescence in the tissue, or have you gotten any other results with positive control tissue? This application is still covered by our Abpromise guarantee, so I will be happy to send a replacement product or issue a credit or refund if we can't get the antibody to work as it should.

I look forward to hearing from you. Please let me know if you have any further questions or if there is anything else that I can do for you.

Staining mouse gut frozen sections, fixed with MeOH or HistoChoice. Primary antibody 1:20 or 1:50. Sees only background, no specific signal. What fixative and primary antibody dilution would be best?

ANSWER:

Thank you for your call yesterday and for letting us know about the trouble with ab8556 in immunohistochemistry.

I apologize for the delay getting back to you,as I'm still waiting for more information from the lab regarding the suggested fixative and dilution for IHC on frozen tissue sections. As we discussed, antigen retrieval can be performed on frozen tissues, and we recommend the article by Yamashita, et al for a good method-

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Yamashita%20S%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlusDrugs1, http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=Search&Term=%22Okada%20Y%22%5BAuthor%5D&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVAbstractPlusDrugs1. Application of heat induced antigen retrieval to aldehyde fixed fresh frozen sections. http://www.jhc.org/. 2005 Nov;53(11):1421-32.

Theyautoclaved tissue sections in pH 9 Tris Hcl for 10 minutes at 120C. Ithink the increased pressure helps the tissue adhere to the slides.

I've also attached a great guide that I found regarding autofluorescence, the various sources, and ways to fix the problem. Theauthorsdon't really discuss glycine treatment but there are several other recommendations that would be helpful.

I will get back to you as soon as I have more information from the lab. I am sorry for the delay, but please let me know if you have any questions in the meantime or if there is anything else that I can do for you.

Thank you very much. I'm curious if when ordering our first vial, we need to enter the discount code? Our previous email stated that this code must be obtained before purchasing the antibody. Thank you!

ANSWER:

Thank you for contacting me regarding this. It is important to have the code prior to ordering the first vial and to include that code as a reference in either your fax or email order, or to tell the customer service representative the code. They will use that to reference the Abreview when you place the subsequent order. Please let me know if you have any questions.