Journal
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
Volume 49, Issue 3, Pages 279-283Publisher
SAGE PUBLICATIONS LTD
DOI: 10.1177/002215540104900301
Keywords
immunogold; silver enhancement; pre-embedding labeling; double labeling; electron microscopy
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Funding
- NICHD NIH HHS [P01 HD35576] Funding Source: Medline
- NINDS NIH HHS [R01-NS35255] Funding Source: Medline
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Pre-embedding double immunogold-silver labeling using two ultrasmall gold conjugates has not been attempted previously because a means of distinguishing labels by conjugates of identical sizes was lacking. This study investigated the feasibility of creating a particle size segregation between two ultrasmall gold conjugates through sequential immunogold incubations and silver enhancements. Two primary antibodies, mouse anti-synaptophysin and rabbit anti-glial fibrillary acidic protein (GFAP), were used in the model system. Differentiation of the double labeling was achieved by incubating with one ultrasmall gold conjugate, followed by silver enhancement, and then incubating with the second ultrasmall gold conjugate, followed by additional silver enhancement. This resulted in two groups of silver-enhanced particles: smaller particles enhanced once and larger particles enhanced twice. Electron microscopic examination revealed two readily distinguished populations of gold-silver particles within the appropriate structures, with very little size overlap. The quality of the ultrastructure permitted identification of most subcellular organelles. This procedure provides for the first time a pre-embedding immunogold-silver labeling protocol that allows the precise subcellular co-localization of multiple antigens.
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