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We generally use the Vector Laboratories ABC kits for IHC. General protocol is as follows (minus wash steps) 1. Frozen sections (mouse spleen) are fixed in cold acetone for 15 min. 2. Endogenous peroxidase blocked with H2O2 in methanol. 3. Blocking stem using VectastainABC kit (for this antibody, normal goat serum) for 30 min RT. 4. Primary staining done at 1/100-1/10,000 dilution using CD11c (N418, from Ab33483) in 0.1% BSA/PBST overnight at 4 degrees. 5. Secondary staining using goat anti-armenian hamster-biotinylated (ab5744) 30 min RT. Tried 1/200, 1/300 in PBST w/ diluted goat serum. Also tried 1/300 in 2% normal mouse serum (abcam). 6. Vectastain ABC reagent (peroxidase), 30 min RT. Without primary, my secondary stains what seems to be the marginal area around the germinal centers.
Asked on May 31 2012
Thank you for this information.
I have noted that this primary/secondary antibody combination has been used and reviewed by another customer. Their Abreview includes a detailed protocol which you may wish to review. This review is at:https://www.abcam.com/CD11c-antibody-N418-ab33483.html&tab=abreviews&intabreviewid=14724
It may be in your case that much of the background is caused by secondary antibody binding to
endogenous mouse IgG in the tissue being stained, and to Fc receptors on B cells, plasma cells and macrophages. As such it may be valuable to use some of the staining procedures for mouse on mouse staining. Blocking endogenous IgG may be done by incubating the sections with an unconjugated Fab fragment likeGoat F (ab')2 polyclonal Secondary Antibody to Mouse IgG - (Fab')2, pre-adsorbed (ab98754),after the normal blocking step.More details may be found at this link:
I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.
Answered on May 31 2012