Open AccessThe Importance of Titrating Antibodies for Immunocytochemical Methods
Author(s) -
Hoffman Gloria E.,
Murphy Kelley J.,
Sita Luciane V.
Publication year - 2016
Publication title -
current protocols in neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.307
H-Index - 40
eISSN - 1934-8576
pISSN - 1934-8584
DOI - 10.1002/cpns.1
Subject(s) - staining , antiserum , immunocytochemistry , antibody , computer science , immunofluorescence , biology , pathology , medicine , immunology
Abstract When using immunocytochemistry, investigators may not know how to optimize staining or how to troubleshoot the method when staining fails. Lacking are guides for comparing techniques and applying information derived from one staining method to another. Newer methods amplify signal detection, but will not necessarily work at the same primary antibody concentrations used for less sensitive reactions. Recommendations of optimal titers are often not accurate and are not usually accompanied by information on the method used to test those antibodies or the specifics of the assay. When the staining does not work, the investigators do not know how to determine if the antiserum is bad, the tissue is bad, or the method is inappropriate for their staining. This unit describes detailed procedures for determining optimal staining and applying that information to three common immunofluorescence methods. Lastly, a formula is provided for converting among the different methods. © 2016 by John Wiley & Sons, Inc.