Biorevo bz 9000 all in one fluorescence microscope

The mPFC and amygdala were identified as region of interest (ROI) based on common histological landmarks (Paxinos and Watson, 2006 ). We used randomization of location and orientation within navigation windows to sample within the mPFC and amygdala. We sampled from infralimbic and prelimbic cortices of the mPFC. Within the amygdala, the anterior basolateral amygdaloid nucleus (BL), the posterior basomedial amygdaloid nucleus (BM), and the ventromedial lateral amygdaloid nucleus (L) were analyzed. c-Fos-positive cells were counted within an unbiased counting frame using a cell count software (Keyence Corp. of America, Itasca, IL, United States). A Keyence Biorevo BZ-9000 All-In-One Fluorescence Microscope (Keyence) equipped with a Nikon CFI Plan Apo λ20X objective (Nikon, Melville, NY, United States) was used to collect z-stacks (numerical aperture, 0.75; working distance, 1.0 mm). Digitalization and image quantification were carried out by blinded observers. Cells identified as c-Fos positive after threshold correction were quantified automatically using the Macro Cell Count Software (batch image analysis tool from Keyence). For immunofluorescence analyses, a minimum of five images per area per animal were used (depending on the size of the ROI). Image analyses were averaged per ROI, and the total sample number used for statistical analysis equaled the number of animals used.

A549 cells (1 × 10⁴ cells/well) were incubated in each well of a 24-well plate with the most potent compounds in this series at IC₅₀ concentrations for 15 h. Then, an apoptotic/necrotic cell detection kit (PromoKine, Heidelberg, Germany) was used according to manufacturer′s instructions with some modifications [43 (link),44 (link)]. Briefly, the cells were washed twice with 1× binding buffer, a staining solution containing 50 μL of 1× binding buffer, 5 μL of FITC-Annexin V solution, and 5 μL of ethidium homodimer III solution, and treated for 30 min at room temperature in a protected-light environment. After washing of cells in 1× binding buffer, cells were analyzed under a Biorevo BZ-9000 all-in-one fluorescence microscope (Keyence, Osaka, Japan). The number of apoptotic cells, late apoptotic or necrotic cells, and necrotic cells was quantified as previously described [45 (link)].