Plan apochromat 20x 0.8 na

For quantification, imaging was conducted using a Zeiss LSM 710 inverted confocal laser-scanning microscope (Carl Zeiss, Jena, Germany) with a 20x objective (Plan-Apochromat 20x/0.8 NA, Carl Zeiss, GmbH, Germany). The boundary of each nucleus of the SCN was determined using Hoechst staining. Receptor density measurements were undertaken using ImageJ (version 1, 50I, National Institutes of Health, Bethesda, MD, USA). After background subtraction (correcting for uneven illuminated background) and gray scale threshold determination (correcting for saturated pixels), the GABA_AR subunit density measurements were performed on each unilateral SCN and these values were averaged (across the left and right nuclei) to produce a single density value. These measurements were expressed with respect to the size of the SCN to obtain an integrated SCN density before averaging across tissue sections (with a minimum of three tissue sections measured per animal) and within treatment groups. Integrated SCN density was normalized to the range of density values in a given immunohistochemistry run for each GABA_AR subunit. The experimenter was blinded to the experimental groupings to eliminate any bias during experiment, including image acquisition and analysis.