A re-evaluation of gelsolin at ectoplasmic specializations in sertoli cells: the influence of serum in blocking buffers on staining patterns.
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.
Guttman
Julian A
JA
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
Vaid
Kuljeet S
KS
Vogl
A Wayne
AW
D000818:Animals; D000906:Antibodies; D016477:Artifacts; D015153:Blotting, Western; D002021:Buffers; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods* / Q000592:standards; D005629:Frozen Sections; D018260:Gelsolin / Q000032:analysis* / Q000276:immunology; D006041:Goats; D007365:Intercellular Junctions / Q000737:chemistry*; D008297:Male; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000737:chemistry*; D044967:Serum / Q000737:chemistry*
D000906:Antibodies; D002021:Buffers; D018260:Gelsolin