Biomedical Engineering Reference
In-Depth Information
Fig. 1. Schematic diagram illustrating typical immunoperoxidase steps of immuno-
histochemistry. After the binding of a primary antibody to its target antigen, the
biotinylated secondary antibody is added to bind to the primary antibody. Through
avidin, the biotinylated horseradish peroxidase binds to the biotinylated secondary
antibody. Finally, horseradish peroxidase catalyzes DAB to give rise to visible brown
reaction products.
of brain sections from living animals or culture slides. This chapter describes
a detailed protocol of immunohistochemistry employed on fl oating brain sections
and immunocytochemistry on culture slides in this laboratory in detecting
pCaMKII, pCREB, pERK, and pElk-1 immunoreactivity in rat or mouse
striatal neurons
(5-8)
. According to the protocol, brain sections (30-40 µm)
cut through the striatum on a freezing sliding microtome or a vibratome or
culture striatal neurons are fi xed with a fi xative, usually 4% paraformaldehyde,
to preserve tissue morphology and the protein in its native site. The sections or
cultured neurons are then permeablized, and nonspecifi c sites are preblocked
with normal animal serum from the species in which the secondary antibody
was raised. Striatal neurons are incubated with the primary antibody to
allow the antibody to bind to the enzyme or factor, followed by incubation
with the secondary antibody conjugated to biotin. After the formation of
antigen-primary antibody-secondary antibody complex, avidin-biotinylated
horseradish peroxidase is added. The peroxidase substrate diaminobenzidine
(DAB) is fi nally added as a chromagen to produce a visible reaction product, an
indirect indication of the phosphorylation of the protein tested (
Fig. 1
).
2. Materials
2.1. Animals and Equipment
1. Adult male or female rats (weighing 200-250 g) or mice (20-25 g) from
commercial vendors. For immunostaining on cultured cells, primary striatal
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