Cx43 microscope

The intracellular reactive oxygen species (ROS) production was examined with dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, Burlington, MA, United States) and fluorescence microscopy (Liu et al., 2010 (link)). The A. alternata hyphae and conidia were treated with S. lydicus M01 extracts for 6 h, centrifuged, and resuspended in 20 mM phosphate buffer (pH 7.5). The samples were then incubated with 20 μM DCFH-DA for 20 min at room temperature and viewed using Olympus CX43 microscope.

Cells were grown on YPD plates for 2 days at 25 °C and the colonies were photographed with an Olympus SZ61 stereo microscope equipped with a CAM-E3CMOS6.3 camera using the ImageView software, and the size measurement performed was using the 3-point circle tool. For the collection of images of the cells, phase contrast microscopy was performed using a Zeiss Observer Z1 inverted microscope Ph3 stage with the Pln Apo 100X/1.4 objective by immobilizing cells from an overnight culture in a 1 mm layer of 1% agarose in PBS. Cells were photographed using the Hamamatsu ORCA-Flash4.0 LT + C11440 camera and the ZEN software. For cell measurement, the cells were photographed with an Olympus CX43 microscope equipped with a CAM-E3CMOS6.3 camera using the ImageView software, and the size measurement was performed with the 5-point ellipsis tool. All figures were prepared with Adobe Illustrator.

Animal adult tissues (around 6 months old) were collected and fixed in 10% neutral-buffered formalin for approximately 24 hours, before being processed and embedded in paraffin. Embedded tissues were cut into 3- to 4-μm sections. Haematoxylin and eosin (H&E) staining was performed at the Histopathology Facility of the IRB, using the standard protocol. Images were acquired using an Olympus CX43 microscope. The histopathological analysis of the adult mice was performed by the Histology Services of the IRB.

For light microscopic analysis, the kidneys were fixed in 10% formaldehyde solution, treated with graded alcohol concentrations for dehydrate, cleared with xylene, and embedded in paraffin. The sections with 4–6 μm thicknesses obtained from renal tissue blocks were stained with hematoxylin and counter stained with eosin. Images of the sections were taken using a ToupCam XCAM 1080PHB camera mounted on an Olympus CX43 microscope. Kidney histopathological changes caused by exposure to fipronil in rats were evaluated using scale of none (0), mild (1), moderate (2), and severe (3) damage.

Following euthanasia, necropsy was performed, gross lesions were noted, and left lungs were harvested in 10% formalin for histopathological assessment. After a 24-h incubation at 4 °C, lungs were transferred to fresh 10% formalin for an additional 48-h incubation before removal from containment. Tissues were processed by standard histological procedures by the UTMB Anatomic Pathology Core, and 4-μm-thick sections were cut and stained with hematoxylin and eosin. Sections of lungs were examined for the extent of inflammation, type of inflammatory foci, and changes in alveoli/alveolar septa/airways/blood vessels in parallel with sections from uninfected or unvaccinated lungs. The blinded tissue sections were semiquantitatively scored for pathological lesions using the criteria described in Supplementary Table 1. Examination was performed with an Olympus CX43 microscope at magnification 40X for general observation and 100X magnification for detailed observation. Each section was scored independently by two trained lab members, and as scores were in agreement, only one set is presented.

All canine tissues were fixed with 10% neutral buffered formalin, decalcified using 12% EDTA with a pH of 7.2, and subsequently embedded in paraffin. An antibody panel against specific immune cell markers (Supplementary Tables 1, 2) was used to stain a subset of 20 specimens. Labeling of canine tissues for CD204 was completed by the Animal Health Diagnostic Center at Cornell University. The remaining canine tissue IHC was completed by the Histology Laboratory at the University of Georgia, College of Veterinary Medicine. Immunohistochemical labeling was examined at 400x (hpf; 0.196 mm²) using an Olympus CX43 microscope. Cells expressing Iba1 and CD204 were numerous and scored semi-quantitatively as 1+ (0–50/hpf), 2+ (51–100/hpf), or 3+ ( >100/hpf). Cells expressing CD3, CD45RA, FOXP3, CD20, and MUM1 were similarly scored as 1+ (<1/hpf), 2+ (1-5/hpf), or 3+ (>5/hpf). Toluidine blue staining was completed by VitroVivo (Supplementary Fig. 10) using the VitroView Toluidine Blue Stain Kit (VB-3013). Two canine tumors were excluded from the immunohistochemical analysis due to poor tissue quality and high background labeling. Five human OS samples, decalcified with formic acid, were labeled and examined for expression of CD3, CD20, and CD204 (Supplementary Table 2).