Nanozoomer scanner

Masson’s trichome staining and Sirius Red staining were used to evaluate the deposition of adipose tissue and collagen in the myocardium. Blood was removed from heart samples, and then the samples were cut transversely into 5 µm slices, fixed in 4% paraformaldehyde solution and embedded in paraffin for staining after deparaffinization and rehydration according to standard procedures [45 (link)]. The stained sections were scanned with a NanoZoomer scanner (Hamamatsu Photonics, Japan) and analyzed with NanoZoomer Pathology Digital software NDPview2.0. The Masson’s trichrome- and sirius red-stained areas were analyzed with Fiji ImageJ software. Slices were randomly selected for analysis under a microscope, and six samples from each group were utilized for analysis.

Stainings were performed on sections from brain tissues fixed in formalin and treated with concentrated formic acid to inactivate prions. Partially protease-resistant prion protein deposits, astrogliosis and microglia deposition were visualized by staining brain sections with the SAF84 antibody (1:200, SPI bio), GFAP (1:1000, Millipore) and IBA1 (1:2500, WAKO) respectively on a NexES immunohistochemistry robot (Ventana instruments) using an IVIEW DAB Detection Kit (Ventana), after preceding incubation with protease 1 (Ventana). Images of DAB stained sections were acquired using the NanoZoomer scanner (Hamamatsu Photonics) and NanoZoomer digital pathology software (NDPview; Hamamatsu Photonics). Quantifications of IBA1, GFAP staining and vacuoles in mouse sections were performed on acquired images; regions of interest were drawn on a Digital Image Hub (Leica Biosystems) and analyzed as previously described [77 ].

Right ovaries were collected from female mice (used for biodistribution) on D15 and D180 and were preserved in 10% buffered formalin and then in 70% ethanol. Whole ovaries were processed in Peloris automaton (Leica) and then embedded in paraffin.
For each animal, 10 sections (3- to 5-μm thick) were manufactured and deposited on two Superfrost+ slides. One slide was treated with the AAV probe, which was designed on the BGH poly(A) region. The second slide was processed according to the same protocol without the AAV probe, which was the negative control. Each assay included positive controls (i.e., a slide of ovary labeled with a control mouse probe designed on the sequence of elongation factor 1 [EF1] and a slide of injected mouse liver labeled with the AAV probe).
Slides were scanned at ×20 magnification in bright-field conditions with a Nanozoomer scanner (Hamamatsu). The analysis consisted of the detection of a positive signal in the slide labeled with the AAV probe by comparison with the negative control slide. The RNA in situ hybridization analysis was performed by Histalim.

The stained slides were scanned into high-resolution digital images at ×20 magnification using a Nanozoomer scanner (Hamamatsu Photonics, Welwyn Garden City, UK). KANK1 cytoplasmic immunoreactivity was evaluated using the modified H-score taking the staining intensity and percentage of positivity into account. Staining intensity (0–3) was multiplied by the proportion of tumour cells (0–100) stained with each intensity and final scores were obtained, giving a range of 0–300 [22 (link)]. Double scoring was assessed blindly by two researchers to evaluate the inter-observer concordance. Intraclass correlation coefficient (ICC) concordance between both observers was 0.9.

Following the slaughter of broiler chickens, the NO and SM breast muscles selected through visual observation and touch, and the fascia and epidermis at the top of the left pectoralis major muscle were removed. Samples of the pectoral muscle tissue, aligned parallel to the muscle fibers (2 cm × 1 cm × 0.2 cm), were fixed in 4% paraformaldehyde fixative (Solarbio) for 48 h. Frozen samples were cross-sectioned and stained with hematoxylin and eosin (Khaliq, Beijing, China, 2023). Subsequently, samples were scanned using a Nano Zoomer scanner (Hamamatsu, Sydney, Australia). Images were acquired under the same conditions and magnification. Four fields of view were obtained per section, and three sections were acquired per sample. We used a microscope to observe the morphology of NO and SM muscle fibers.

Lungs and spleens were formalin fixed, ethanol washed, and paraffin embedded. Sections were obtained and stained with H&E, using standard techniques. Slides were scanned at 20X using a Hamamatsu Nanozoomer scanner and images obtained from the scanned slides at magnifications indicated by the scale bars. All slides were interpreted by a Board Certified Veterinary Pathologist (Dr. Meegan Larsen, MBed Pathology).