Post-embedding double-labeling of antigen-retrieved ultrathin sections using a silver enhancement-controlled sequential immunogold (SECSI) technique.
In electron microscopy, the post-embedding immunogold technique provides a high degree of resolution and the possibility of quantitation owing to the intrinsic characteristics of the colloidal gold marker. Application of this technique to the subcellular localization of multiple antigens by differential labeling using gold markers of different sizes, or to double labeling using the same primary antibody isotype with serial silver enhancement, has been reported. We have incorporated this double labeling technique into a modified procedure that produces excellent labeling and ultrastructural preservation, even after exposure of ultrathin sections large enough to cover a 300- micro m-diameter single-hole grid to hot antigen retrieval solutions and prolonged labeling protocols.
Goode
Nigel P
NP
Renal Research Unit, St James's University Hospital, The Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom. n.p.goode@leeds.ac.uk
Shires
Michael
M
Crellin
Doreen M
DM
Khan
Tahir N
TN
Mooney
Andrew F
AF
D000185:Actinin / Q000378:metabolism*; D016616:Cryoultramicrotomy; D000939:Epitopes; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D019012:Integrin beta1 / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000378:metabolism*; D016622:Silver Staining
D000939:Epitopes; D019012:Integrin beta1; D008840:Microfilament Proteins; C503967:SYNPO protein, human; D000185:Actinin