Ds fi1 digital camera

For microscopic analysis the material was fixed in 70% ethanol. Preliminary observations of the morphology of roots (thickness, branching, presence of root hairs) were examined using a Nikon SMZ1000 stereoscopic microscope. Afterwards, the roots were hand sectioned and subjected to anatomical investigations by bright-field light microscopy (LM) and fluorescence microscopy using a Nikon Eclipse Ni-U microscope equipped with a Prior 200W lamp (Prior Scientific Instruments Ltd., Cambridge, UK) and UV-2B cube filter 355/50 (330–380 nm excitation filter; a 400 nm (LP) dichroic mirror and a 435 nm (LP) barrier filter) to detect autofluorescence of cell walls. Additionally, hand-cut sections were stained for general histology using 0.05% Toluidine blue O [48 ]. Micrometry and photomicrography were carried out using a DS -Fi1 digital camera (Nikon, Japan) and NIS-Elements BR software.

Immunocytochemistry was performed to determine the composition of the bladder cell wall. The tissue section on the slide was incubated in blocking solution (1% bovine serum albumin in phosphate buffer saline; pH 7.2; PBS-BSA) at 37°C for 1 h and then with diluted (1:10 in BSA) CCRC-M1 (recognizes fucose (Fuc)-containing epitope), CCRC-M7 (arabinogalactan epitope), and CCRC-M38 (de-esterified homogalacturonan) primary monoclonal antibodies (Carbosource Services, Georgia, United States) for 1 h. After incubation, the slides were washed five times with PBS-BSA and allowed to react with horseradish peroxidase (HRP)-labeled secondary antibody (goat anti-mouse IgG) diluted in PBS-BSA (1:500) at 37°C for 1 h. In control, the primary antibodies were replaced with PBS. Finally, all sections were stained with diaminobenzidine (DAB; ZSGB-BIO, ZLI-9018) for 3 min and dehydrated twice in absolute ethanol and xylene for 5 min. The sections were observed under a Nikon ECLIPSE Ti-E microscope, and the digital images were captured with a Nikon DS-Fi1 digital camera; the extended focal range images were analyzed and visualized with the NIS-Elements D imaging software (version 4.0).

Paraffin-embedded liver sections were stained by the PAS reaction and counterstained with haematoxylin in a Leica immunostainer. Sections were visually evaluated and imaged on a DMRB microscope (Leica, Wetzlar, Germany) using a DS-Fi1 digital camera (Nikon Instruments, Düsseldorf, Germany) controlled by the NIS Elements BR software. Following automated PAS-quantification is described in detail in Supplementary material and methods.
Of 34 liver samples with PAS intensities covering the full range from weakly stained to strongly stained, glycogen content was determined biochemically using the Glycogen Assay Kit II (ab169558; Abcam, Cambridge, UK), according to the manufacturers protocol to measure glycogen.

Immunohistochemical analysis was performed on cryosections taken from the center of each wound [2 (link),7 (link),33 (link)]. Sections were air-dried, fixed in cold acetone, washed with PBS, quenched with 0.3% hydrogen peroxide, and washed with PBS. Sections were blocked with buffer containing 3% bovine serum albumin and then incubated with F4/80 antibody to label macrophages (1:100, eBioscience, San Diego, CA, USA) or Ly6G antibody to label neutrophils (1:100, BD Pharmingen, San Diego, CA, USA). Sections were then washed with PBS and incubated with biotinylated anti-rat secondary antibody (1:200, Vector Laboratories, Burlingame, CA, USA). After a wash with PBS, sections were incubated with avidin D-horseradish peroxidase (1:1000) and developed with a 3-amino-9-ethylcarbazole kit (Vector Laboratories). Digital images were obtained using a Nikon Instruments Eclipse 80i microscope with a 20x/0.75 objective, a DS-Fi1 digital camera, and NIS Elements software. The percent area stained in each image was then quantified as described above for trichrome and CD31.

After successful bradyzoites isolation, parts of skin samples (5 × 5 mm²) were stored in 10% phosphate-buffered formalin for histopathological examinations. Shortly, formalin-fixed samples were dehydrated using an ascending ethanol series, embedded in paraffin wax at 56°C and finally sectioned at 3 μm tissue samples at the Institute of Veterinary Pathology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Germany. Histological tissue samples have been stained using haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Giemsa staining according to routine protocols and pathological findings/changes of B. besnoiti-infected skin samples were then evaluated under a light microscope (Nikon Eclipse 80i) equipped with a DS-Fi1 digital camera (Nikon).