FITC Anti-alpha smooth muscle Actin antibody [1A4] (ab8211)

Thank you very much for your question. Indeed, the use of ab8211 in flow cytometry has been documented in the publication "Delayed Goblet Cell Hyperplasia, Acetylcholine Receptor Expression, and Worm Expulsion in SMC-Specific IL-4Rα–Deficient Mice " PMID 17222057 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1769405/?tool=pubmed "For the intracellular stain, cells in single cell suspension were incubated with 1% normal rat serum and stained with rat anti-mouse IL-4Rα–PE (mIL-4RM-1; BD Biosciences, http://www.bdbiosciences.com). Stained cells were then washed, fixed in 2% paraformaldehyde, permeabilized with saponin, preblocked with 2% NRS and Fc block (2.4G2), and stained with anti–α-actin FITC (Abcam, https://www.abcam.com) or isotype control IgG2a-FITC (BD Biosciences). " I hope this helps the customer. In conjunction with the protocol on intracellular flow cytometry https://www.abcam.com/index.html?pageconfig=resource&rid=11448, this should allow the reproduction of good results. Please let me know if the customer needs more information. Please let me also know if the customer has any problems. I would be pleased to investigate this for him. As you know, we do cover all our products to work as stated on the datasheet. I wish you a great week.

Thank you for your email. Indeed, I can confirm that the protocol is in reference to ab8211. The use of blocking is to decrease unspecific signal and background. The Fc block with the monoclonal antibody 2.4G2 is to block the Fc receptors expressed on certain cells, such as macrophages. The Fc receptors have a high affinity for immunoglobulins and might therefore bind the primary antibody via its Fc chain- resulting in an unspecific staining. The 2.4G2 is directed against Fc receptors and is shown to block their ability to bind immunoglobulins. The additional use of NRS (normal rat serum) is to block unspecific protein-protein interaction. It is therefore dependent on the cell type the customer is analysing whether these two blocking methods are necessary. It is however very good practice to include them and I can recommend to do so. I hope this information is helpful. Please do not hesitate to contact me again should the customer have other questions.