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Immunofluorescence staining is the use of a fluorescent antibody
(i.e. antibody conjugated with a fluorochrome) for the detection and/or quantification
of a specific antigen (or antibody). In the simplest immunolourescence techniques,
used to detect antigen, the specimen (tissue section, smear, etc) is exposed
to the conjugate (i.e. dyelinked antibody) for an appropriate time, washed free
of conjugate, and examined by fluorescence microscopy; antigens homologous to
the fluorescent antibodies are readily identified by regions of fluorescence
in the specimen.
In the indirect immunofluorescence technique, the presence of
a given antigen can be determined by first exposing the specimen to unlabeled
antibodies; the specimen is then washed free of unbound antibodies, exposed
to fluorescently labeled anti-Ig antibodies, washed, and examined by fluorescence
microscopy. Any antigen-antibody combination on the specimen can be detected
by the presence of fluorescent antibodies (which bind to unlabeled antibodies);
if particular antigens are known to be present in the specimen, the test can
be used to detect homologous antibodies in the serum.
Confocal fluorescence microscopic examination to demonstrate
the expression and distribution of E7 and chimeric FL-E7 proteins. 293 Db,Kb
cells were transfected with pcDNA3-E7-GFP (A–C) or pcDNA3-FL-E7-GFP DNA
(D–F) using Lipofectamine. Immunofluorescent staining was performed as
described in "Materials and Methods." For the detection of GFP protein,
green fluorescence was noted (B and E). For the detection of endogenous calnexin
protein, red fluorescence was observed (A and D). Controls omitting primary
antibodies did not demonstrate specific red fluorescence (data not shown). Colocalization
of GFP and calnexin was demonstrated by the yellow color in the combined image
(C and F). (Enhancement
of DNA Vaccine Potency by Linkage of Antigen Gene to a Gene Encoding the Extracellular
Domain of Fms-like Tyrosine Kinase 3-Ligand)
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