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A re‐evaluation of gelsolin at ectoplasmic specializations in sertoli cells: The influence of serum in blocking buffers on staining patterns
Author(s) -
Guttman Julian A.,
Vaid Kuljeet S.,
Vogl A. Wayne
Publication year - 2007
Publication title -
the anatomical record: advances in integrative anatomy and evolutionary biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.678
H-Index - 62
eISSN - 1932-8494
pISSN - 1932-8486
DOI - 10.1002/ar.20441
Subject(s) - gelsolin , staining , antibody , monoclonal antibody , immunohistochemistry , actin , chemistry , frozen section procedure , microbiology and biotechnology , intracellular , biology , pathology , immunology , medicine
In this study, we test the hypothesis that gelsolin immunolocalized in actin filament‐rich ectoplasmic specializations may be exogenous gelsolin present in normal serum used in blocking buffers, and that binds to the intercellular adhesion plaques during tissue processing. Fixed frozen sections of rat and rabbit testis were pre‐treated with standard blocking buffers containing 5% normal goat serum (NGS) and then incubated with anti‐gelsolin antibodies in the presence of 1% NGS. Other sections were treated in a similar fashion, but in buffers not containing NGS. Sections were then labeled with secondary antibody conjugated to a fluorochrome. Localized staining at ectoplasmic specializations occurred only in sections treated with NGS. The only positive staining in sections not treated with NGS was associated with seminiferous tubule walls and blood vessels in rabbit tissue. The antibodies reacted with a single band at the appropriate molecular weight for gelsolin on immunoblots of NGS, but did not react on immunoblots of testis or seminiferous epithelium. We conclude that gelsolin localized at ectoplasmic specializations using current commercially available antibodies is a result of non‐specific binding to the fixed tissues of gelsolin present in blocking buffers. Anat Rec 2007. © 2007 Wiley‐Liss, Inc.