Dm lb2 microscope

We imaged and counted 3 rostrocaudal sections per region in the right hemisphere (+3.2 to +2.88 mm for the mPFC and −2.7 to −3.02 mm for the amygdala; DM LB2 microscope, Leica Microsystems, North Ryde, Australia) as described previously (Kim et al., 2012 (link)). Because DARPP-32 levels did not vary across any groups, DARPP-32/c-Fos double labeling was standardized as a percentage of total DARPP-32 immunostaining (%DARPP-DBL). Images from the mPFC were cropped as individual prelimbic (e.g., PrL) or IL regions, and amygdala images were cropped into subregions, central amygdala (e.g., CeA), basal amygdala (BA) and lateral amygdala (LA) for manual counting, which were delineated into standardized area size according to the rat brain atlas (Paxinos and Watson, 1998 ). Observers unaware of experimental groups counted using ImageJ (NIH, MD, USA).

The bacterial membrane integrity was assessed by fluorescence microscopy using the Live/Dead (LD) BacLight^® kit. This methodology uses a mixture of two dyes (SYTO-9 and Propidium iodide [PI]) that allows the assessment of the membrane integrity by selective stain exclusion [70 (link)]. For this purpose, 300 μL of biofilm suspensions were filtered through a Nucleopore^® (Whatman International Ltd., Maidstone, UK) black polycarbonate membrane (pore size 0.22 μm) and stained with LD dyes, according to the manufacturer’s instructions. Afterwards, the samples were observed in a DMLB2 microscope (LEICA Microsystems Ltd., Weltzlar, Germany) equipped with a HBO/100W/3 mercury lamp. Images were captured using a CCD camera and IM50 software (LEICA). The percentage of green (stained with SYTO-9) and red cells (stained with both SYTO-9 and PI) was quantified from 20 microscopy fields. Controls included dark and irradiation treatments.