Stereological Quantification of Tyrosine Hydroxylase Positive Cells

Stereological probes were applied using a Zeiss Imager M2 microscope (Carl Zeiss, Jena, Germany) equipped with StereoInvestigator software (v2019.1.3; MBF Bioscience, VT, USA) according to previously published methods [37 (link), 38 (link)]. Using the optical fractionator probe, Tyrosine Hydroxylase (TH) positive cells were counted in sections 480 μm apart using a grid size of 170 X 100 μm and counting frame size of 75 X 75 μm. Contours were drawn around the region of interest at 10X magnification, and cells were counted under a 63X oil immersion objective. Guard zones were set at 2 μm each at the top and bottom of the section, and the counting frame was lowered at 1-2 μm interludes and each cell in focus was marked. The Gundersen method for calculating the coefficient of error was used to estimate the accuracy of the optical fractionator results. Coefficients obtained were generally less than 0.1.