Quantitation of tyrosine hydroxylase protein in the locus coeruleus from postmortem human brain.
In this study, we developed an immuno-autoradiographic method to obtain quantitative estimates of tyrosine hydroxylase (TH) protein in tissue sections from post-mortem human brain. Protein from tissue sections containing the locus coeruleus (LC) was directly transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoreactive TH on PVDF membranes was identified with optimized concentrations of TH antibody followed by application of [125I]labeled secondary antibody. Quantities of TH on autoradiograms were estimated by comparing optical densities of transferred immunoblots to a calibrated standard curve produced with purified recombinant TH dotted onto the same PVDF membranes. Amounts of TH-immunoreactivity in the LC were proportional to the thickness of tissue sections up to 15 micrometer. However, the amounts of total protein, as measured by Ponceau S staining, were linearly related to section thicknesses up to 30 micrometer. Comparisons of quantities of immunoreactive TH in the LC using this method to amounts determined using traditional Western blotting, in which LC tissue was punched from adjacent sections from the same subject, showed a positive correlation (r(2)=0.99, P<0.01). Using the transfer immunoblot method, an uneven distribution of TH protein was observed along the rostrocaudal axis of the human LC (P<0.01). This method may provide a sensitive and useful tool for the study of the role of human TH expression in the pathophysiology of psychiatric disease.
Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, 2500 North State Street, 39216-4505, Jackson, MS, USA.
M
M
Zhu
V
V
Klimek
J W
JW
Haycock
G A
GA
Ordway
D001345:Autoradiography / Q000379:methods*; D015153:Blotting, Western; D006801:Humans; D007163:Immunosorbent Techniques; D008125:Locus Coeruleus / Q000166:cytology / Q000201:enzymology*; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis*
D014446:Tyrosine 3-Monooxygenase