Optical densitometry of tyrosine hydroxylase fibers
mariangela.massarocenere
Abstract
Protocol for quantifying TH+ fibers in rat striatum and substantia nigra pars reticulata (SNpr)
Steps
Acquire images with a light microscope of immunostained serial coronal sections covering the caudo-rostral of the brain regions (3sections/animal for striatum and SNpr)
Open ImageJ (RRID:SCR_003070,https://imagej.net/)
Calibrate grey mean values for standard optical density values
Open 8-bit images in ImageJ, trace the region of interest (striatum, SNpr), and measure the optical density. Then, in the same image, trace the region of interest in a blank area (cortex for striatum, surrounding neuropil for SNpr ) and measure the optical density.
Calculate the optical density by subtracting the background optical density from the striatal optical density (if traced region of interest areas are the same) or by the formula: SNpr Integrated density-[(SNpr area) x (background optical density)]
