Axiovert 200

Cell concentrations and chain lengths were analyzed every second or third day in Sedgewick-Rafter counting chambers on a Zeiss Axiovert 200 microscope.
Morphological analysis was performed at the end of the exponential growth phase. Five subsamples of 20 μL for each culture were analysed by DAPI staining on a Zeiss Axioimager fluorescence microscope using the Zeiss filterset 49 for epifluorescence as well as bright field phase contrast for transillumination^{30 (link)}. Only cultures with no bacterial sized (0.2–3 μm) and DAPI positive particles were further analyzed. Morphological analyzes were carried out using Zeiss Axiovert 200 light microscope (LM) (Carl Zeiss, Oberkochen, Germany) equipped with Nomarski differential interference contrast (DIC), phase contrast, and bright-field optics. Light micrographs were taken using a Zeiss Axiocam digital camera and all morphological measurements were made in software suite Axiovision 4.8 (ZEISS, Oberkochen, Germany). The terminology used to describe morphological features of Leptocylindrus species follows Anonymous³¹ and Ross et al.³² . Biovolume of Leptocylindrus species was calculated using the following formula: V = π/4 * cell diameter²* cell height^{33 (link)}.

Quantification of ataxin-3 positive inclusions was performed blindly by scanning 4 coronal sections spread over the anterior-posterior extent of the cerebellum (inter-section distance: 240 µm), using a 20× objective on a Zeiss Axiovert 200 imaging microscope and an image acquisition and analysis software (ImageJ, NIH). For each coronal section and animal 8 fields were acquired, covering the same areas. The average number of inclusions in the cerebellum was calculated for each animal. The quantification of DARPP-32 and calbindin neuronal expression was performed blindly by scanning 4 coronal sections spread over the anterior-posterior extent of the cerebellum expressing the mutant ataxin-3 protein (inter-section distance: 240 µm), using a 40× and 20× objective on a Zeiss Axiovert 200 imaging microscope. For each coronal section and animal, 8 fields were acquired using the same exposure and covering the same areas. Optical densitometry analysis was performed using Image analysis software (ImageJ, National Intistute of Health, U.S.A). Cerebellar regions expressing DARPP-32/calbindin revealed to have an increased optical density value when compared to regions not expressing the protein. Values are represented as the mean value of DARPP-32/Calbindin optical density per section ± SEM (arbitrary units).

Macrophages (2 x 10⁵) were incubated in a petri dish overnight. The morphology of the macrophages attached to the dish was observed under a microscope with a 20X or 40X objective lens (Axiovert 200, Carl Zeiss, Inc.) at Ewha Fluorescence Core Imaging Center. Morphological differences were enumerated in 3 different fields in each sample, and the percentage of cells well spread (or the cellular area) was calculated. The area of each individual cell was determined using Image J analysis software. Macrophages (1 x 10⁵) attached on a cover glass were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. After being blocked with 1% BSA for 1 hour, the samples were incubated with FITC- or rhodamine-conjugated phalloidin for 1 hour and then with DAPI for 5 minutes at room temperature. After being mounted, the samples were analyzed using a Zeiss Axiovert 200 inverted microscope that was equipped with an X40 Achroplan LD or X20 Plan-Neofluar objective, a Zeiss AxioCam HRc, and an Axio Vision Rel. 4.8 software (Carl Zeiss, Oberkochen, Germany).