Fluorescent indirect immunocytochemistry

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

Fig. 7.2 Indirect immunocytochemistry. An unlabeled 1° antibody binds to the antigen and a labeled 2° antibody binds to the 1° antibody. The labeled 2° antibody is made in a different species of animals against the IgG species of the 1° antibody. A variety of labels can be used from fluorescence to enzymes...

Offers modest increase in sensitivity over indirect immunocytochemistry with fluorescent-labeled 2°. 

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

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).

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