Tyrosine hydroxylase immunohistochemistry and tyrosine hydroxylase-immunoreactive cell count

Tyrosine hydroxylase immunohistochemistry
and tyrosine hydroxylase-immunoreactive
cell count
Fifty-five days after surgery, the animals
were sacrificed and the midbrains were
placed in 4% formalin for 1 week and then in
20% sucrose formalin for 48 h before sectioning.
The brainstems were frozen and
series of 30-μm thick sections were cut with
a sliding microtome on the frontal plane.
One series of sections was immunostained
for tyrosine hydroxylase (TH) with a monoclonal
anti-TH antibody raised in mice
(Incstar Corp., Stillwater, MN, USA) at a
1:5000 dilution. The primary antiserum was
located by applying a variation of the avidinbiotin
complex system using a commercially
available kit (ABC Elite kit, Vector Laboratories,
Burlingame, CA, USA) as described
in a previous study (6). The number of THimmunoreactive
neurons was determined
using a 10X objective of a Nikon Eclipse
E600 microscope equipped with a camera
lucida by counting these neurons on both
sides of the SNc and ventral tegmental area
in 8 series of coronal sections located 360
μm apart, and the estimated counts were
extrapolated using the method of Abercrombie
(10). The borders defining the SNc and
ventral tegmental area were determined from
adjacent Nissl-stained sections that served
as a reference for cytoarchitectural purposes.