Axiovert 200m microscope

The right lower lobe of the liver was harvested from infected mice and fixed in 4% buffered formalin, embedded in paraffin, and then sectioned at 5 to 7 μm. The liver sections were stained with hematoxylin and eosin (H&E) to reveal granulomas. All the histopathological images were captured at 100× magnification using Axiovert 200M microscope coupled with a digital camera (Zeiss, Oberkochen, Germany).
The liver sections were stained using the Masson’s Trichrome staining kit (Sigma-Aldrich) for semi-quantitative analysis of hepatic fibrosis. Six to eight fields from each slide were randomly obtained at 100× magnification using Axiovert 200M microscope coupled with a digital camera (Zeiss). The blue-stained area per total area and the fibrosis intensity were determined with Image-Pro Plus software (Media Cybernetics, Rockville, MD). The total fibrosis density score was determined by dividing the image intensity by the image area. Intensity exclusion parameters were identical for all the captured images.

Images of tumor, lymph node and kidney sections were captured with the following microscopes: A stereo-fluorescence microscope MZ16 FA (Leica) equipped with a digital CCD camera and the Leica IM1000 4.0 software (1300×1030 pixel RGB-color images), a TCS SP2 AOBS confocal laser microscope (Leica) equipped with the LCS 2.16 software (1024×1024 pixel RGB-color images) and a Axiovert 200 M microscope (Zeiss) with Axiovision 4.5 software (1388×1040 pixel gray scale images), respectively. Digital images were processed with Photoshop 7.0 (Adobe Systems, USA) and merged to yield pseudo-colored pictures. Images of cells seeded in 24 well plates were captured either with the stereo-fluorescence microscope MZ16 FA (Leica) or with the Axiovert 200 M microscope (Zeiss).

For microscopic analysis a Zeiss Axiovert 200M microscope (Zeiss, Oberkochen, Germany) equipped with a motorized stage (Märzhäuser, Wetzlar, Germany) with MosaiX software and by means of a CCD camera (Zeiss MRC) connected to an AxioVision 4.8.2 image analysis system (Zeiss) was used to create tile pictures. Each tile picture is composed of 285 single microscopic images taken at a magnification of 50 and represents a whole well of a 12-well plate. As denoted in the figure legend to Fig. 2 ImageJ images in this figure have been graphically enhanced for representation purposes using the Corel Draw Graphics Suite 2017 (Corel Corporation, Ottawa, Canada).

Tissues were harvested and fixed in 4% paraformaldehyde overnight at 4 °C and subsequently were dehydrated and embedded in paraffin as previously described56 (link). Tissues were rehydrated and stained with hematoxylin and eosin (H&E) according to the manufacturer’s protocol (Sigma–Aldrich, USA). Slides were mounted with Fluka Eukitt quick-hardening mounting medium (Sigma–Aldrich, USA). Images were obtained using a Zeiss Axiovert 200M microscope with a Zeiss FLUAR 10x/0.5 NA objective and PALMRobo V4.3 software (Carl Zeiss, Germany).
The TUNEL assay was performed in accordance with the manufacturer’s protocol (Roche). For immunofluorescence analyses, tissue sections were rehydrated and incubated in 5% normal goat serum (NGS) for 1 h and then incubated with the primary antibodies in 3% NGS overnight at 4 °C. Subsequently, sections were washed and then incubated with respective secondary Alexa Fluor 594-conjugated antibodies (Invitrogen) for 1 hour at room temperature. Slides were then mounted with Hoechst 33342 dye (Life Technologies, USA). Images were obtained using a LSM 710 confocal microscope with a Zeiss EC Plan-NEOFLUAR 20x/0.5 NA objective and were analyzed using ZEN 2012 Blue Edition software (Carl Zeiss, Germany).

Cells were incubated for 24 h with 20 μM CldU. They were then incubated with 20 μM Dig-TMP for 20 min before UVA irradiation in a Rayonet chamber or exposure to the 365 nm laser in ROI in nuclei in cells in the immediate vicinity of the cross. Cells exposed to the UVA lamp were harvested by trypsinization and the cells were placed on a glass slide. Plates with cells containing laser localized ICLs were washed and a drop of trypsin from a drawn out capillary placed on the intersection of the cross. The detached cells were recovered and placed on a glass slide. Cells were then mixed with lysis buffer (0.5% SDS in 200 mM Tris-HCl [pH 7.5], 50 mM EDTA) on the slide. After tilting, the slides were air-dried, fixed in 3:1 methanol/acetic acid, incubated in 2.5 M HCl, neutralized in 0.4 M Tris-HCl (pH 7.5), washed in PBS, and immunostained. Antibodies and dilutions were rat anti-BrdU (CldU), 1:200; Dylight 649 goat anti-rat, 1:100; mouse anti-BrdU (IdU), 1:40 and chicken anti-digoxigenin, 1:200; and Dylight 488 goat anti-mouse, 1:100 and Q dot 655 goat anti-chicken, 1:2,500. Imaging was carried out using a Zeiss Axiovert 200 M microscope with the AxioVision software packages (Zeiss). The quantum dot signal was imaged with a Q dot 655 filter.