Anti-HIF-1 alpha 抗体 [ESEE122] (ab8366)

Thank you for your inquiry.

The placental tissue was a rather unique sample of which we know of no commercial source of and also did not get any more information form the scientist that used it.

For an IHC positive control we recommend using glioblastoma multiformae.

I am sorry I could not be of more help and hope this information is nevertheless helpful.

Thank you for your call yesterday and for your patience while I have been in touch with the lab regarding the cytospin staining protocol for the images shown on the datasheet.

We do not have much more information about the protocol, as the cytospin stains were done by an outside lab in 2005 and we did not receive the full protocol. The primary antibody was conjugated to a fluorophore prior to the staining, so no secondary antibody was used. We do not have information about the how the cytospin was prepared.

I am sorry that we don't have further information regarding this protocol, but if there is anything else that we might be able to do for you please let me know and I'll be happy to help.

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.

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: https://www.abcam.com/hif1-alpha-antibody-esee122-ab8366.html ab1: https://www.abcam.com/HIF1-alpha-antibody-H1alpha67-ChIP-Grade-ab1.html ab1 reviews: https://www.abcam.com/HIF-1-alpha-antibody-H1alpha67-ab1.html&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.

Thank you for your enquiry. I apologize that I am unable to answer you in Spanish. Ab8366 has not yet been tested in Western blot, so it may be this antibody is not suitable for that application. You may wish to submit an Abreview of your results from the antibody datasheet. Plese take a look at ab1 and ab2185, both of which were tested in WB and cross-react with rat. I hope this information helps. Please do not hesitate to contact us if you need anything further.

120 kDa is the expected band but the 60 kDa band is often seen in both hypoxic and normoxic cells. The same band is often seen when using ab1 as well. We do not know what the 60 kDa band represents at this time. However, using nuclear extracts decreases this background band and increases the HIF-1 alpha signal. ***Abcam does not recommend ab8366 in Western blotting ***

Generally speaking, we recommend NB 100-131 (ab8366) for immunohistochemistry work. It has been used for western analyis on human and rat samples (MCF7 and PC12 nuclear extracts). The other antibody of which you speak is ab2185, a polyclonal made to the same fusion protein as ab1 and ab463. It has been tested on the same samples as those mentioned above. ***Update 2006: we do not recommend ab8366 for Western blotting***

The 1:8,000 dilution was done for 90 minutes at RT by some researchers at the University of Oxford with antigen unmasking. It has also been done at 1:1,000 for 40 hrs at 5C (see review for this product) with pre-treatments (Dako). The dilutions and incubation time seem to vary according to the protocol.