Abstract
AbstractProtocols for blocking non-specific antibody (Ab) binding in immunohistochemistry are based on rather contradictory and outdated reports. This prompted us to prove, whether non-specific Ab binding may really lead to unwanted background staining in routinely processed cell and tissue probes. In this study, the probes were fixed and processed according to routine protocols with and without a blocking step (goat serum or BSA). Surprisingly, all Ab in probes processed without a blocking step did not show any propensity towards non-specific binding that might lead to background staining, thus implying that endogenous Fc receptors do not retain their ability to bind Fc portion of Ab after standard fixation. Likewise in routinely fixed probes, we did not find any non-specific Ab binding ascribed to a combination of ionic and hydrophobic interactions. The traditionally used protein blocking step is useless in immunostaining of routinely fixed tissues.
Highlights
The causes of non-specific background immunostaining might be different, but they have one thing in common: they may complicate the use of immunohistochemistry
Omission of incubation with primary Ab in negative controls did not led to unwanted background stainings due to anticipated non-specific binding of secondary Ab in probes processed without the protein blocking step, which means that the protein block traditionally used in immunohistochemistry does not influence the quality of immunostaining
Contrary to the speculative declaration that the unspecific background staining due to endogenous Fc receptors (FcRs) is more common for frozen sections and cell smears than for paraffinembedded tissue sections[19, 20], the unspecific background staining has not appeared to be a problem with frozen tissue sections fixed either with formaldehyde (Fig. 1a-c) or with acetone, as well as with blood cell smears, cell culture monolayers and cytospins fixed in formaldehyde
Summary
The causes of non-specific background immunostaining might be different, but they have one thing in common: they may complicate the use of immunohistochemistry. Other FcRs are expressed on multiple cell types and are similar in structure to MHC class I This receptor is involved in preservation of antibodies and binds IgG3. The most reasonable approach to prevent the possible non-specific background due to FcRs might be the use of F(ab′)[2] fragments of Ab instead of the whole IgG molecule[11], provided that the endogenous FcRs retain their ability to bind Fc portion of IgG Ab after proper fixation, but that is namely the question. Other blocking solutions based on bovine serum albumin (BSA), coldwater-fish gelatin, trypton casein peptone, non-fat dry milk or casein are assumed to prevent non-specific background ascribed primarily to hydrophobic interactions of proteins and to ionic or electrostatic interactions[9, 12, 13]. The list of recommendations of this kind can be extended, but their practicability is questionable and they are rarely - if any at all - used in praxis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
