Anti-HIF1 alpha antibody [ESEE122] (ab8366)

Hi xxxx,
Thank-you so much for your speedy response. I am really looking forward to this new batch as I have a mountain of samples to get through and hopefully publish.
The address that you have supplied is correct … Not sure who Steve Lubb is/was but he is not on our staff data base so just make it for my attention.
I feeling really optimistic. Thanks again.

Best Regards

ANSWER:

Thank you for confirming that information.


I have now organised for a new vial of ab8366 to be sent out to you. You can expect it to arrive tomorrow. The order number is xxxx(PO number FOCR xxxx). If you have any trouble receiving it please do let me know.


I would be very interested to hear how you get on and if you have the time, as mentioned in my previous email, if you could share the details of the protocol you intend to usewith me by filling out the questionnaireI attached to the previous email (and re-attached to this email). Seeing some images of the staining when it worked and when it didn't would also be very helpful in investigating this case further.


Many thanks for your cooperation and I wishyou all the best with your new experiments.

ab is staining the negative tissues also
Samples- Prostate cancer tissue sections and Normal prostate cancer tissue sections
IHC-P application
Microwave antigen retrieval method for 30 minutes.
Dilution 1/10,000
The antibody use to work previously

ANSWER:

Thank you very much for your phone call last week.

HiF1 alpha is a very sensitive target it stabilizes under hypoxic conditions and gets degraded under normoxic conditions. Upon stabilization HIF-1 translocates to the nucleus so we recommend permeabilizing the sections.

I have attached the protocol that we recommend for HIF1 alpha target staining. Although this protocol is not different than others so may be troubleshooting few steps will helps
- Antigen retrieval always needs optimizations. 20 minutes is only a suggested antigen retrieval time for Microwave. Less than 20 minutes may leave the antigens under retrieved, leading to weak staining. More than 20 minutes may leave them over-retrieved, leading to nonspecific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 5, 10, 15, 20, 25 and 30 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
- We would recommend using citrate buffer as antigen retrieval buffer however for optimizations purpose different buffers can be used e.g. EDTA, Tris EDTA.
- Tissues sections: the tissue should be fixed immediately after removal; any delays actually degrades the protein.
- We suggest incubating the section in Normal immune serum or 5% BSA for at least 1-2 hours.
- Following publications may also help with protocol
http://ajpheart.physiology.org/content/289/5/H2066.long
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC468666/?tool=pubmed
Same clone number has been mentioned in following publications
Beasley et al. 2002. Cancer Res. 62:2493-7. PMID: 11980639
Giatromanolaki et al. 2003. J. Pathol. 200:222-8. PMID: 12754744
Hui et al. 2002. Clin Cancer Res. 8:2595-604. PMID: 12171890
I hope these suggestions will help. Should you have any further question please do not hesitate to contact me.

Bonjour Mme ******* de ******* (Maroc) nous a livré deux anticorps que nous avons demandés pour un projet : HIF 1 alpha et CAIX; la boite ne contient que les anticorps concentrés sans diluent et sans anticorps secondaires.Pour le diluent nous pouvons éventuellement utiliser le TBS comme c'est conseillé sur votre site mais pour l'anticorps secondaire qu'elle serait la solution?

ANSWER:

Merci de nous avoir contactés. Les anticorps primaires Abcam sont livrés sans diluent ni anticorps secondaire. J'ai retrouvé la commande passée par ****. Cette commande concerne en effet 2 anticorps primaires, l'anti-HIF1 alpha antibody [ESEE122]référence ab8366 et l'anti-Carbonic Anhydrase IX antibody [SP106]référence ab105226. Le lien vers la liste des anticorps secondaires compatibles avec ab8366 (Mouse IgG1)est : http://www.abcam.com/index.html?pageconfig=searchresults&search=IgG1&pt=7&sk=targsp&sv=2&sn=Mouse&l=0&fViewMore=1 Le lien vers la liste des anticorps secondaires compatibles avec ab105226 (Rabbit IgG) est : http://www.abcam.com/index.html?pageconfig=searchresults&search=IgG&pt=7&sk=targsp&sv=2&sn=Rabbit&l=0&fViewMore=1 Vous pouvez affiner votre recherche en utilisant les filtres à gauche de la liste (application, conjugué, etc). La solution de dilution dépendra de l'application testée. J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions. Bonne et heureuse année 2012 à vouségalement.

I need an anti-HIF antibody that will work in IHC-Fr on mouse tissue. I also need positive controls for this.

ANSWER:

Thank you for contacting Abcam.

I have had a chance to look through the data for at HIF1 alpha antibody panel (ab10625) that you had spoke of during our conversation. Of the antibodies included in this panel only ab8366 (Anti-HIF1 alpha antibody [ESEE122]) has been tested to work in IHC-Fr. Although the other antibodies may work in with this condition they have not been tested as such and therefore I cannot be confident that this product will meet your needs completely.

As such I would recommend using ab8366 for your experiments. Positive controls for this product include glioblastoma multiformae and hypoxia-induced human placenta.

I would also like to highly recommend ab1 (Anti-HIF1 alpha antibody [H1alpha67] - ChIP Grade). This product has been tested in IHC-IF on mouse and has been referenced in 33 publications. The Abreviews for this product may also be an excellent resource for your experiments. I've included links to both these antibodies as well as the direct link to the IHC-Fr Abreviews for Ab1 below.

ab8366: http://www.abcam.com/hif1-alpha-antibody-esee122-ab8366.html

ab1: http://www.abcam.com/HIF1-alpha-antibody-H1alpha67-ChIP-Grade-ab1.html

ab1 reviews: http://www.abcam.com/index.html?datasheet=1&tab=abreviews&intabreviewid=9511

I hope this information helps you. I would also like you to know that these products are covered by our Abpromise®. Which ensures that you can trust our products to perform as stated on the datasheets, or we will offer a replacement, credit, or refund.

The Abcam Abpromise® guarantee: • 100% Scientific and Customer Support for any product you buy from Abcam or one of our authorized distributors. • We guarantee our products work in the tested species and applications stated on the datasheet. • We will replace or refund products not performing as stated on the datasheet if reported within 6 months of purchase. • We investigate any quality concerns raised by customers to ensure our catalog contains products that perform to the highest standards.

Please note these conditions to our Abpromise®: • Protocol information must be provided in order for the claim to be validated. • Antibodies tested in recombinant samples only are not guaranteed for use on endogenous samples. • “Predicted to react” information on the datasheets is provided for reference only and these species are not guaranteed. • Fast Track antibodies are covered for ELISA against the immunizing peptide only. I hope this information is helpful, but please do not hesitate to contact us with further questions.

Anti-HIF1 alpha antibody [ESEE122] (ab8366) has a declared reactivity with cow. Could you direct me to the reference that showed this reactivity or if it is not from published work can you tell me how this reactivity was confirmed?

Kind Regards,

ANSWER:

I am very sorry for the delay, and I really appreciate your patience.

Unfortunately there is no publication available to prove the reactivity of ab8366 with cow. However, as the datasheet states, this product is guaranteed to work with cow species, and it is therefore covered by our guarantee Abpromise, that ensures this product should work in the species and applications indicated in the datasheet.

If you had any problems when testing it in cow, we would be happy to give you scientific support and offer you a free of charge replacement, refund or credit note in the case it does not work.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.