Axio imager a1

S. plumieri venom was injected in the intraplantar region of the hind foot paw of mice (n = 4–7) according to Boletini-Santos et al. [30 (link)]. After 24 h, mice were euthanized with an i.p. pentobarbital injection (60 mg/kg; Sanofi, Libourne, France), the chest wall was opened, and the tracheas were cannulated to obtain bronchoalveolar lavage fluid (BAL). The collected BAL was centrifuged (170× g for 10 min at 20 °C), and the resulting cell pellet was then resuspended in 1 mL of PBS. Cell counts were performed using a hemocytometer, and cytocentrifuge (Cytospin II; Shandon, Cheshire, UK) slides were stained (Hema 3, Scientific Products, Chicago, IL, USA). For differential cell counts, 300 leukocytes were enumerated and identified as macrophages on the basis of staining and morphologic characteristics using a conventional light microscope (Axio Imager A1, Carl Zeiss, Germany). Paraffin-embedded sections of the left lung were stained with hematoxylin and eosin (H&E). All slides were examined with light microscopy at a magnification of 10 or 40× (Axio Imager A1, Carl Zeiss, Germany).

Live T. spiralis muscle larvae were fixed in cold ethanol and xylene and embedded in paraffin for histological analysis. Five-micron sections were deparaffinized in xylene, rehydrated with a graded alcohol series, washed and incubated with rabbit anti-human galectin-1 affinity-purified polyclonal antibody (1:1000) (Institute for the Application of Nuclear Energy - INEP, Belgrade, Serbia), overnight at 4 °C. After washing, sections were incubated with FITC-conjugated anti-rabbit IgG secondary antibody (1:1500) (Molecular probes, Thermo Fisher scientific, Waltham, MA, USA) or with biotin-labeled goat anti-rabbit IgG secondary antibody (1:500) (Vector Laboratories, Burlingame, CA, USA), for 1 h at room temperature (RT). Non-specific binding was assessed by omitting the anti-human galectin-1 incubation step. Indirect immunofluorescence (IIF) staining was examined by ultraviolet microscopy (AXIO Imager A1, Carl Zeiss AG, Goettingen, Germany). Incubation with biotin-labeled goat anti-rabbit IgG was followed by ABC (avidin/biotinylated horseradish peroxidase complex, Vector) (1:1000) for 30 min, and the reaction was developed by adding the chromogenic substrate. The stained sections were viewed using light microscopy (AXIO Imager A1, Carl Zeiss AG).

Liver fragments were fixed in 10% neutral formalin, dehydrated in ascending alcohol solutions, and embedded in paraffin. Sections (4-5 μm thick) were stained using the hematoxylin/eosin techniques and studied using light microscopy (AxioImager A1, Carl Zeiss). Specific histochemical staining by the Ziehl-Neelsen stain was used to visualize Mycobacterium bovis in the tissues. The numerical density of the granulomas and their diameters were determined by using the morphometry method (AxioVision software, rel. 4.8). These parameters were used as the morphological criteria for tuberculosis activity.
For immunohistochemical study, 3 μm sections were dewaxed and dehydrated, and epitopes were retrieved in citrate buffer solution in a 700 W microwave oven for 20-35 minutes. Endogenous peroxidase was blocked. The samples were incubated with primary anti-Nrf2 antibodies according to instructions. Then, the sections were incubated with horseradish peroxidase (HRP) conjugate (using a Spring Bioscience Corporation detection system) and 3,3′-diaminobenzidine (DAB) substrate and then stained with Mayaer's hematoxylin. Sections were dehydrated using increasing concentrations of ethanol and xylol mounted with synthetic mounting media “Bio Mount,” placed under cover glasses and studied using light microscopy (AxioImager A1, Carl Zeiss).