Quantitative autoradiography of tyrosine hydroxylase immunoreactivity in the rat brain.
Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.
INSERM U 289, Hôpital de la Salpêtriére, Paris, France.
R
R
Raisman-Vozari
E
E
Hirsch
F
F
Javoy-Agid
C
C
Vassort
M
M
Savasta
C
C
Feuerstein
J
J
Thibault
Y
Y
Agid
D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D003342:Corpus Striatum / Q000201:enzymology; D007150:Immunohistochemistry; D008297:Male; D008636:Mesencephalon / Q000201:enzymology; D051381:Rats; D013462:Sulfur Radioisotopes; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D013462:Sulfur Radioisotopes; D014446:Tyrosine 3-Monooxygenase