Nonspecific Nuclear Immunoreactivity After Antigen Retrieval Using Acidic and Basic Solutions

Optimization, validation, and identification of two reliable antibodies for immunodetection of WNT5A

“…Although we did not evaluate whether the perinuclear immunostaining pattern was related to the nuclei or it was an artifact as others have reported, we did not observe this immunostaining pattern with the monoclonal 3A4 antibody. If this staining pattern is an artifact that is obtained only with the polyclonal AF645 antibody, it could be due to effects of HIAR on the antigen-antibody binding activity, which is consistent with an earlier interpretation (Wieczorek et al 1997). Regardless of the reason for the perinuclear immunostaining pattern of the AF645 antibody, the phenomenon complicates interpretation of IHC staining for WNT5A.…”

Optimizing immunohistochemistry: antigen retrieval and signal amplification

“…Nucleolar staining using L26 has also been reported 96 . Retitration of primary and/or secondary antibodies will usually abolish the problem 97 , 98 . Endogenous biotin may be unmasked 99 , necessitating stronger blocking procedures.…”

Technical Aspects of Immunohistochemistry

“…The cause of this nonspecific binding is unclear, but it has been hypothesized that a combination of electrostatic and polar (electron acceptor/electron donor) forces are involved in this problem. 11, 159 Detection of some Ags is improved if the ionic strength of the fixative solution is increased. 17 Solving nonspecific background staining in immuno-histochemistry becomes more complicated with the realization that, many times, this nonspecific staining is the result of a combination of ionic and hydrophobic interactions, and, as previously mentioned, remedies for one type of interaction can aggravate the other.…”