Eclipse ni upright microscope

The mouse macrophage cell line RAW 264.7 was seeded at 6 × 10⁴ cells per well in a 24-well plate containing a 10-mm microscope cover glass (Glaswarenfabrik Karl Hecht, Sondheim vor der Rhön, Germany). The cells were infected with bacteria carrying pBSK-Km:ZsGreen in DMEM-glucose medium containing IPTG (0.5 mM) and kanamycin (50 µg/mL) for 1 h. After washing twice with PBS, the cells were incubated in PBS containing 20 μg/mL gentamycin and 50 μg/mL streptomycin for 30 min to eliminate residual extracellular bacteria. Subsequently, cells were fixed in 3% paraformaldehyde at room temperature for 30 min and then permeabilized with 0.1% Triton X-100 in PBS. After washing with PBS, the cells were counterstained with CF^®647 phalloidin (Biotium, Fremont, CA, USA) to stain F-actin and DAPI (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) to identify the nuclei. A 10-mm microscope cover glass with cells was placed and mounted on a microscope glass slide for observation by a Nikon C2Si laser confocal microscope configured with a Nikon Eclipse Ni upright microscope (Nikon, Tokyo, Japan) at a magnification of 600×. Confocal images were analyzed using 64-bit NIS-Elements AR version 4.60.00 imaging software (Nikon, Tokyo, Japan).

Cells plated on glass coverslips were permeabilized with 50 µg/ml digitonin in PBS for 10 min at RT. Cells were then washed three times with PBS and incubated in blocking solution (2% BSA in PBS) for 15 min at RT. After three washings with PBS, cells were incubated with 1 µg/ml lysenin in blocking for 2 h at RT. Cells were then washed three times with PBS and incubated with rabbit lysenin antiserum (1:500 in blocking solution, 1 h at RT). Cells were washed three times with PBS and incubated with anti-rabbit AlexaFluor488 antibody (1:600 in blocking solution, 45 min at RT). After PBS washes, coverslips were mounted on a glass slide with Dako fluorescent mounting medium. The fluorescence emitted by Lysenin was detected using a NikonEclipse Ni upright microscope (400 × magnification) and a Nikon DIGITAL SIGHT DS-U1 CCD camera. Images were analyzed and fluorescence intensity was quantified using the ImageJ software. At least 20 fields were examined for each sample.

All mice, either upon sacrifice or upon reaching the end of the study, underwent necropsy. The primary tumor site, the left lung of each mouse, and any sites of obvious extrapulmonary metastatic disease were harvested and fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (HE). Bony tissues were decalcified in 10% formic acid. Stained sections were visually reviewed using a Nikon Eclipse Ni upright microscope (Nikon Instruments Inc., Melville, NY) by a board-certified veterinary pathologist.