Axio imager a2 epifluorescence microscope

Raw lake water and the 5.0 μm pre-filtered filtrate were fixed with formaldehyde (a final concentration of 2%) and used to enumerate the abundances of total bacteria (TB) and free-living bacteria (FLB), respectively. TB and FLB were enumerated by a flow cytometer according to Gong et al., 2017 (link). Then, the abundance of particle-attached bacteria (PAB) was calculated as TB minus FLB. For the abundance of detrital particles (Tang et al., 2009 (link)), the fixed raw water samples were stained by 4′,6′-diamidino-2-phenylindole (DAPI, Sigma) at a final concentration of 2 μg/ml for 10 min, filtered onto black polycarbonate filters (0.2-μm pore size, 25-mm diameter, Poretics)^TM and enumerated using Axio Imager A2 epifluorescence microscope (Zeiss, Germany) equipped with a high speed, high resolution and sensitivity camera (Hamamatsu, Janpan). At least 100 particles (diameter > 5 μm) were counted and be photographed. The acquired particle images were analyzed by HCimage live software. Then the abundance of particles, mean diameter and mean area of particles in each lake were calculated.

The metaphase and polytene chromosomes were obtained from neuroblasts and salivary glands of third instar larvae of D. virilis (strain 15010–1051.51) and D. americana (strain H5), according to [44 (link),45 ]. Probe labeling and FISH experiment conditions were conducted according to [16 (link)]. The satDNA probes were immunodetected with antidigoxigenin-Rhodamine and avidin-FITC (Roche Applied Science).
We used DAPI “4,6-diamidino-2-phenylindole” (Roche) in “SlowFade” antifade reagent (Invitrogen) for DNA counterstaining. The analyses were conducted under an Axio Imager A2 epifluorescence microscope equipped with the AxiocamMRm camera (Zeiss). Images were captured with Axiovision (Zeiss) and edited in Adobe Photoshop.

Resected tumors were frozen on dry ice in a cryo-embedding compound (Ted Pella, Inc; 27300), sectioned, and fixed in a 3:1 mixture of acetone:ethanol. Non-specific binding was blocked with 2% BSA in PBS, followed by staining with an antibody mix containing rabbit anti-mouse Ki67 antibody (Cell Signaling Technologies; 12202) and rat anti-mouse CD31 antibody (Dianova; DIA-310). Detection of primary antibodies was achieved using FITC conjugated goat anti-rabbit IgG (BD Pharmingen; 554020) and Cy3 conjugated goat anti-rat IgG (Invitrogen; A10522). Samples were counterstained with DAPI (Vector; H-1500) and mounted with a hard-set mounting medium for fluorescence. Random images from each section were obtained with a Zeiss AxioImager A2 epifluorescence microscope at 200x magnification, and analyzed with ImageJ. CD31+ cells (% area) and Ki67+ cells (% cells) were quantified automatically using macro functions, whereas Ki67+/CD31+ cells (proliferating endothelial cells) were quantified manually.