St5020 autostainer

After noting the placement site, the catheter segments were removed from the brains, which were then processed and embedded in paraffin wax using standard laboratory protocols (PathCentre Tissue Processor: 3 day schedule). The neuropathologist carrying out the assessment (I. S. S.) was blind to group allocations. Sections were stained using haematoxylin and eosin (Leica ST5020 autostainer), and with antibodies raised against glial fibrillary acidic protein (GFAP) (Roche) (at a dilution of 1/5000), β-amyloid precursor protein (β-APP) (Roche) (1/20000) and neurofilament protein (NFP) (Roche) (1/50). Immunohistochemistry was performed on a Ventana Benchmark Ultra (Roche Diagnostics) automated stainer. The slides were dewaxed using cell conditioner 1 (Roche) at 95°C for 36 min and the primary antibodies were applied as follows: 24 min (GFAP), 32 min (NFP) and 36 min (β-APP). Haematoxylin counterstain was applied for 12 min. The slides were then mounted prior to viewing (Thermo Scientific ClearVue). Negative controls were performed by omitting the primary antibody. Positive controls were mounted on all slides having first demonstrated cross-reactivity between human and mouse tissues.

Blood, liver, and kidney were collected upon euthanasia. Tissues were fixed in 4% paraformaldehyde for 24 hours and then placed in 70% ethanol at 4°C for 4 to 6 days before paraffin embedding. Sections were cut at 5 μm and stained with hematoxylin and eosin on the Leica ST5020 Autostainer, and whole slide scanning was performed by a Leica AT2 slide scanner at ×20 magnification. Slides were reviewed by two veterinary pathologists using HALO Link software (Indica Labs) and Leica DM 3000 LED microscopes.

Five-micron sections from formalin-fixed paraffin-embedded papilloma blocks were deparaffinized and rehydrated using the Leica ST5020 Autostainer. Antigen retrieval was performed using a Leica Bond citrate-based solution (Leica AR9961). Single color automated staining was performed using anti-human CD4 (Abcam ab133616; 1:400 dilution) or CD8 (Abcam ab182729; 1:200) antibodies applied for 60 min. After washing, secondary antibody staining and colorimetric development was perfumed using the Leica BOND Polymer Refine Detection kit (#DS9800) per manufacturer recommendations. Coverslipping was performed with the Leica Automated Coverslipper (#CV5030). All samples were stained in the same batch with appropriate positive (tonsil tissue) and negative controls. Images of stained slides were acquired on a Vectra Polaris using brightfield illumination. Analysis of images was performed using QuPath. Percentage positivity of stained cells for entire FFPE sections was calculated using common detection thresholds for all samples.