Fluorescent immunocytochemistry

Fig. 17. Drawings showing the relative expression levels of AMPA receptor subunit GluR-A to -D mRNAs in parvalbumin- (PV) and calretinin- (CR) immunopositive cells of the adult rat hippocampus (non-radioactive in situ hybridization combined with indirect fluorescence immunocytochemistry). Each dot represents one cell. Intensity levels are black, strongly positive grey, moderate or lightly labelled white, negative (Catania et al., 1998).

Keywords Immunohistochemistry Antibody labeling Fluorescence microscopy Fluorescent immunocytochemistry Fluorescent immunohistochemistry Indirect immunocytochemistry Immunostaining... 

The r antibody incubations for samples are the same as those used for fluorescent immunocytochemistry, with labeled 2° antibodies instead labeled with small gold. The first step is a permeabilization and blocking step, followed by two rinses. The detergent selected will affect the quality of the morphology observed. The strongest detergent is Triton X-100 at 0.3%, although some authors have used upto... 

Aqueous mounting media - a solution miscible with buffers, does not need dehydration, and is used with fluorescence immunocytochemistry. 

Fluorophore - a fluorescent molecule that is excited by a wavelength of light and emits a higher wavelength of light used in fluorescent immunocytochemistry... 

In contrast, when using fluorescence immunocytochemistry to label proteins in tissue sections, the absolute relationship of fluorescence intensity to protein abundance can only be determined through conducting rigorous control experiments, and thus linearity should not be presumed. With that said, because CCD and photomultipHer tube detection systems are linear across a broad range of fluorescence intensities [36], quantitative fluorescence microscopy has been used in numerous smdies to measure relative changes in protein level [33, 35, 36]. In a system where the expression of a protein is only effected in a subpopulation of cells (e.g., GAD67 expression is reduced in a subset of PV+ interneurons in schizophrenia—see ref. 45), quantitative fluorescence immunohistochemistry will be more informative than techniques (e.g.. Western blot) that treat the tissue as a whole. 

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