Single cell analysis of the expression of a nuclear protein, SCIP, by fluorescent immunohistochemistry visualized with confocal microscopy.
A widely applicable method for the accurate quantification or semiquantification of macromolecules at the level of individual cells is described and validated; this is a method which may considerably facilitate the study of many biological processes. This method relies on measuring fluorescent emission in immunocytochemically labelled cells with a confocal microscope. Emission is related quantitatively to the level of the fluorophore by the combination of an analysis of the polarization of the fluorescent emission and fluorophore rationing methods. The method was applied to the study of the expression of the suppressed cyclic AMP-induced POU protein (SCIP) transcription factor in glial cells of the central nervous system. In particular, the method allowed the study of transcription factor expression in defined cells present in heterogeneous cultures and in cell types which cannot be isolated in sufficient numbers for biochemical analysis using conventional techniques.
Bögler
O
O
Neurobiology Laboratory, Ludwig Institute for Cancer Research, London, UK.
Entwistle
A
A
Kuhn
R
R
Monuki
E
E
Lemke
G
G
Noble
M
M
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D009457:Neuroglia / Q000378:metabolism*; D050816:Octamer Transcription Factor-6; D051381:Rats; D017208:Rats, Wistar; D012583:Schwann Cells / Q000378:metabolism; D013194:Staining and Labeling; D013234:Stem Cells / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology
D009419:Nerve Tissue Proteins; C494179:Pou3f1 protein, rat; D014157:Transcription Factors; D050816:Octamer Transcription Factor-6; D016650:Fluorescein-5-isothiocyanate