Dfc490

Quantification was performed by systematically sampling each selected region using counting frames superimposed over the region with a microscope (Leica Microsystems DFC490, Germany) coupled to a computer with the Leica Application Suite X software (Leica Microsystems, Germany) with a total magnification of 192X. The sizes of the counting frames were 250,000 μm for CG, PL and IL and 72,000 μm for CA1, CA3 and DG. The total area sampled by these frames per region in each section was: 500,000 μm for CG, PL and IL; 144,000 μm for CA1 and CA3 and finally, 72,000 μm for DG. c-Fos-positive nuclei were defined based on homogenous gray-black stained elements with a well-defined border. Finally, the mean c-Fos positive nuclei count in two sections was calculated for each subject and region.
The regions of interest and their distances (mm) counted from bregma according to the atlas of Paxinos and Watson (2007) were: +3.24 mm for the cingulate cortex (CG), prelimbic cortex (PL) and infralimbic cortex (IL) and -3.24 mm for the CA1, CA3, and the dentate gyrus (DG) sub-fields of the dorsal hippocampus. In these regions, we quantified the number of c-Fos positive nuclei in two alternate sections 30 μm apart. The slides were coded so that the researcher who performed the entire analysis did not know the treatment of the individual subjects.

Histochemical GUS staining was performed as described previously (Urano et al. 2004 (link)), and GUS was observed using an M205C stereomicroscope equipped with a DFC490 digital color camera (Leica Microsystems). To determine the subcellular localization of TCP13, plasmid DNA (5 μg) isolated from 35Spro::TCP13-sGFP plants was transfected into Arabidopsis mesophyll protoplasts, and GFP was detected under a confocal laser scanning microscope (LSM510; Zeiss). Arabidopsis protoplast isolation and PEG-calcium transfection were performed as described previously (Yoo et al. 2007 (link)).

After culture, NR explants were fixed for 2 hours in 4% paraformaldehyde (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and processed in an automatic tissue processor (Leica ASP300; Leica Microsystems, Wetzlar, Germany). One paraffin block per NR was made and 5 μm thick sections were obtained with a microtome (Microm HM340E; Microm International GmbH part of Thermo Fisher Scientific, Walldorf, Germany).
For NR histological characterization, sections were dewaxed in xylene, rehydrated in a series of descending alcohols, rinsed in deionized distilled water, and stained with hematoxylin and eosin (H-E) (Sigma-Aldrich®). Stained sections were dehydrated in a series of ascending alcohols, cleared in xylene, mounted, and cover-slipped. NR samples were analyzed with a light microscope (DM4000B; Leica Microsystems), and images were acquired with a digital camera (DFC490; Leica Microsystems). Brightness and contrast were minimally adjusted, and the final figures were composed with Pixelmator 3.8 Phoenix (Pixelmator Team, Vilnius, Lithuania).