Nis elements f

Immunostaining of cardiac tissue was performed as previously described^{25 (link)}. To analyze the cell death in mouse heart samples, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using Click-iT®TUNEL Alexa Fluor® kit according to manufacturer’s protocol (Invitrogen, C10246). Briefly, deparaffinized samples were incubated with TdT enzyme at 37 °C for 1 h and signals were developed with reaction buffer (Alexa Fluor 594) for 30 min in dark room. After wash the samples with 3% bovine serum albumin (BSA) solution, nucleus was stained with Hoechst 33342 and analyzed with confocal microscopy. For immunocytochemistry, cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min. Cells were then blocked with blocking buffer (2% BSA in 0.1% PBST) for 30 min and incubated with primary antibodies diluted in blocking buffer for overnight at 4 °C. Images were analyzed with an LSM-710 confocal microscope system (Carl Zeiss), Tissue FAXS i8 plus (TissueGnostics) or Nikon ECLIPS TE-2000U and processed with ZEN software (Carl Zeiss), TissueQuest analyzer (TissueGnostics), NIS-Elements F (Nikon) or image J software.

Blood was collected by venipuncture of the jugular or cephalic veins of dogs into EDTA and clot tubes for hematology and serum biochemistry, respectively. Stained blood smears were examined by oil immersion light microscopy at 1000× magnification using the Nikon Eclipse N-U microscope, fitted with the Nikon DS-Ri1 camera (Nikon Corporation, Tokyo, Japan) operated by the NIS-Elements F software package (Nikon Corporation, Tokyo, Japan). Parasites were measured and photographed. All measurements are in micrometres and are given as the range followed by the mean ± standard deviation (SD) in parentheses.

The microstructure analysis was conducted following Hernández-Carrión et al. [37 (link)], with some modifications. A Nikon ECLIPSE 80i light microscope (Nikon Co., Ltd., Tokyo, Japan) was used working in the bright field and fluorescence modes. The autofluorescence of the phenolic compounds in the samples was observed using a mercury arc lamp with a tetramethyl rhodamine filter (λ_ex = 543/22 nm, λ_em = 593/40 nm) as the excitation source. Samples were visualized using 10× and 20× objective lenses. The images were captured and stored at 1280 × 1024 pixels using the microscope software (NIS-Elements F, Version 4.0, Nikon, Tokyo, Japan).

A manual inverted microscope (Ti-S; Nikon, Tokyo, Japan) equipped with a Super Plan Fluor ELWD ADM 20/0.45 was used to observe the eGFP expression in mycelia, conidia, germ tube, appressorium and invasive hyphae. eGFP was exited using a HG fiber illuminator with filter cube (Excitation Filter EX450-490, Dichroic Mirror DM505, Barrier Filter BA520, Tokyo, Japan) and images were acquired using a high-resolution color camera head DS-Ri2 (Nikon, Tokyo, Japan). All parts of the system were under the control of the NIS-elements F (Nikon, Tokyo, Japan).

For histological analysis, samples of conjunctiva, both freshly explanted and after treatment at 37 °C for 5 h with the different blank vehicles reported in Table 2, were fixed in 10% formaldehyde. Subsequently, the samples were embedded in paraffin and sectioned using a microtome. Staining was carried out using Harris hematoxylin/eosin. Images were taken using an optical microscope Nikon Eclipse 80i, equipped with a camera Nikon Digital Sight DS-2Mv and connected to the control software, NIS Elements F (Nikon Instruments, Calenzano, Italy).