Axiovert 135 tv microscope

Bright field images of capsules were captured with a Zeiss Axiovert 135 TV microscope. Images were taken using either a ×2.5 or a ×10 objective. In some cases, the microscopy was performed with slight under-focus, which helped to clearly define the outlines of the layers and/or the overall capsule.

Immunofluorescence analysis was performed exactly as described previously (28 (link)). Samples were collected at times indicated, washed with phosphate-buffered saline (PBS), and fixed either with paraformaldehyde (4%) followed by permeabilization with 0.5% Triton X-100 or by methanol (5 min at −20°C). Samples were blocked with PBS containing 10% NCS or a mix of 5% goat serum, 5% NCS, and 2% bovine serum albumin (BSA), for 1 h at room temperature. Primary antibodies for the antigens were anti-ICP4 antibody (Virusys) used at 1:400 and anti-ICP8 antibody used at 1:400. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma), and coverslips were mounted in Mowiol supplemented with 2.5% 1,4-diazabicyclo(2,2,2)octane (DABCO). Images were taken using an Axiovert 135 TV microscope using Zeiss 10×, 40× LD, or 63× lenses (Plan-Apochromat, 1.4 numerical aperture) and captured using a Retiga 2000R camera with Image Pro Plus software or with a Zeiss laser scanning confocal microscope (Zeiss Pascal).

All the samples processed for optical microscopy were visualized and photographed with an Axiovert 135 TV microscope (ZEISS, Germany) equipped with an Olympus DP50 digital camera and a fluorescence lamp. The images were obtained with the program ViewFinder^TM 7.1 (Better Light, USA) and processed with the program Adobe PhotoShop CS4 EXTENDED V11.0 (Adobe systems, USA).

Cell counting was performed as described previously [30 (link)]. mHSCs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS; Sigma-Aldrich, Taufkirchen, Germany) and incubated with Hoechst 33342 (Fluka, Buchs, Switzerland) for 5 min. Microscopy was performed with a Zeiss Axiovert 135 TV microscope with a Zeiss AxioCam MRm camera (objective lens Fluar 10×/0.50), using Zeiss AxioVision SE64 Rel. 4.9 imaging software. Nuclei count was performed with ImageJ software (Version 1.53c, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Gametophytes were allowed to develop for 10.5–16 h and viewed by placing several drops of culture media containing emerging and fully developed spermatozoids in a shallow dish with a coverslip for a bottom. Spermatozoids were analyzed using differential interference contrast (DIC) microscopy with a Zeiss Axiovert 135 TV microscope. Movies were taken with a PIKE F032B monochrome camera (Allied Vision Technology) using StreamPix software at 30 frames per second. Movie files were converted to .mov format. Still frames from movies were captured and converted into tiff format for analysis.

The level of DNA damage was assessed by the alkaline Comet assay (Singh et al., 1988 (link)). Liver and kidney samples from all experimental groups were minced in ice-cold HBSS buffer (0.14 g/l CaCl₂, 0.4 g/l KCl, 0.06 g/l KH₂PO₄, 0.1 g/l MgCl₂ × 6H₂O, 0.1 g/l MgSO₄ × 7H₂O, 8.0 g/l NaCl, 0.35 g/l NaHCO₃, 0.09 g/l Na₂HPO₄ × 7H₂O, 1.0 g/l D-glucose) containing 20 mM EDTA and 10% DMSO. Ten microliters of a liver or kidney cell suspension was mixed with low-melting agarose (0.75%) and applied to a microscope slide. Cells were lysed for 2 h at 4°C in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% Triton X-100). After lysis, the slides were incubated for 30 min at 4°C in electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH 13.0) and subjected to electrophoresis in order to separate the damaged DNA fragments. The slides were placed in neutralization buffer (0.4 M Tris-HCl, pH 7.4) and stained with SYBR Green I (1:10,000 dilution; Sigma–Aldrich). Fluorescence microscopy was performed with a Zeiss Axiovert 135TV microscope equipped for epifluorescence. DNA damage was quantified by measuring the displacement between the genetic material contained inside the nuclear sphere or comet ‘head’ and the resulting comet ‘tail.’ Images were analyzed with TriTekCometScore Freeware v1.5.¹