TY - JOUR
T1 - Antigen unmasking for immunoelectron microscopy
T2 - Labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium
AU - Stirling, J. W.
AU - Graff, P. S.
PY - 1995/2/1
Y1 - 1995/2/1
N2 - To optimize the ultrastructural localization of immunoglobulin G in corneal crystalloid deposits, we compared a range of antigen unmasking techniques. A human corneal biopsy specimen was fixed in formalin, post- osmicated, and embedded in epoxy resin for electron microscopy. Thin sections were immunogold-labeled for IgG after treatment with sodium ethoxide or sodium metaperiodate. Sections were also treated by heating them at 95°C while they floated on water, 0.01 M citrate buffer (pH 6.0), or sodium metaperiodate. The treatments were applied separately and combined. After labeling, crystalloids in untreated sections had a probe density of 5 particles/μm2. Crystalloids in sections treated only with sodium ethoxide or sodium metaperiodate had probe densities of 15-20 particles/μm2. Sodium ethoxide combined with heating on water, or citrate buffer, gave probe densities of 140-160 particles/μm2. Sodium metaperiodate combined with heating on titrate buffer gave the highest probe density (195 particles/μm2). Although sodium ethoxide coupled with heating increased probe density, the ethoxide etched the sections and caused unacceptable damage. Treatment with sodium metaperiodate followed by heating on citrate buffer is recommended for antigen unmasking. This combination gave a high probe density and sections remained intact, with good ultrastructural detail.
AB - To optimize the ultrastructural localization of immunoglobulin G in corneal crystalloid deposits, we compared a range of antigen unmasking techniques. A human corneal biopsy specimen was fixed in formalin, post- osmicated, and embedded in epoxy resin for electron microscopy. Thin sections were immunogold-labeled for IgG after treatment with sodium ethoxide or sodium metaperiodate. Sections were also treated by heating them at 95°C while they floated on water, 0.01 M citrate buffer (pH 6.0), or sodium metaperiodate. The treatments were applied separately and combined. After labeling, crystalloids in untreated sections had a probe density of 5 particles/μm2. Crystalloids in sections treated only with sodium ethoxide or sodium metaperiodate had probe densities of 15-20 particles/μm2. Sodium ethoxide combined with heating on water, or citrate buffer, gave probe densities of 140-160 particles/μm2. Sodium metaperiodate combined with heating on titrate buffer gave the highest probe density (195 particles/μm2). Although sodium ethoxide coupled with heating increased probe density, the ethoxide etched the sections and caused unacceptable damage. Treatment with sodium metaperiodate followed by heating on citrate buffer is recommended for antigen unmasking. This combination gave a high probe density and sections remained intact, with good ultrastructural detail.
KW - Cornea
KW - Electron microscopy
KW - Human
KW - Immunogold
UR - http://www.scopus.com/inward/record.url?scp=0028911485&partnerID=8YFLogxK
U2 - 10.1177/43.2.7529784
DO - 10.1177/43.2.7529784
M3 - Article
C2 - 7529784
AN - SCOPUS:0028911485
SN - 0022-1554
VL - 43
SP - 115
EP - 123
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 2
ER -