Aperio scanscope cs

Four μm paraffin-embedded tissues sections were retrieved on and incubated with antibodies against tryptase (DAKO, Glostrup, Denmark) and CD31 (Novocastra, Leica Biosystems, Nussloch, Germany) according to manufacturers’ instructions. The slides were then incubated with biotinylated anti-mouse Igs, peroxidase-conjugated streptavidin and diaminobenzidine (DAB). Counterstaining was performed with hematoxylin. Ten slides were scanned for each antibody using the whole-slide scanning platform Aperio Scanscope CS (Leica Biosystems, Nussloch, Germany), then were assessed with the Positive Pixel Count algorithm embedded in the AperioImageScope software and reported as a percentage of positivity, defined as the number of positively stained pixels on the total of pixels of the image.

Adjacent tissue sections for each tissue were stained with hematoxylin and eosin for general histology. For immunohistochemistry, polyclonal antibodies for B. mallei were generated in-house. Briefly, rabbits were immunized with 400 μg of formalin-killed whole cell B. mallei. Two booster immunizations of 400 μg were given 1 month and 4 months after the primary immunization. Rabbits were bled 2 years after the initial immunization and the antibody containing serum was isolated. FFPE tissue sections were placed on superfrost plus slides (Thermo), deparaffinized, rehydrated, blocked with methanol hydrogen peroxide for 30 minutes, and rinsed in phosphate-buffered saline (PBS), pH 7.4, for immunohistochemistry. Serum-free CAS-Block (Life Technologies, Grand Island, NY) containing 5% goat serum (Vector Labs, Burlingame, CA) was applied to the slide for 30 minutes. The polyclonal B. mallei antibody was diluted 1:500 in PBS and incubated with the tissue (60 min, 20°C). After washing, polymer labeled horseradish peroxidase anti-rabbit secondary antibody (Dako, Carpinteria, CA) was applied for 30 minutes, rinsed, counter stained with hematoxylin, dehydrated, and cover-slipped with Permount (Thermo, Hampton, NH). Whole slide images were scanned for analysis and manipulation using an Aperio Scanscope CS (Leica Biosystems) to assess lesions.

H&E and Oil-Red O histochemical staining on frozen muscle sections was performed as previously described by Sullivan et al. [40 (link)]. Images were acquired using the Aperio ScanScope CS (Leica Biosystems) at × 20 magnification and visualised using the Aperio Image Scope companion software (version 11.2.0.780, Lecia Biosystems). The Aperio Image Scope ‘Colour Deconvolution v9’ algorithm was used to analyse the percentage of red stained (Oil-Red O positive) areas in each muscle section. However, it must be noted that Oil-Red O staining on cryosections is confounded by smearing of lipids across the section, which makes it difficult to quantify and to determine whether lipid droplets were localised in myofibers or adipocytes [17 (link)]. To help overcome this caveat, the staining intensity and area of Oil-Red O-stained areas was accounted for to calculate the staining index.