Aperio cs2 slide scanner

Hematoxylin–eosin (HE) staining was performed using the previous method (21 (link)). Specifically, the liver and spleen tissues were fixed with 4% paraformaldehyde, dried with gradient alcohol, cleared with dimethylbenzene, and then embedded in paraffin. A rotary microtome (Leica, Germany) was then used to slice the tissues into 5 μm thin slices. The tissue sections were inspected under a microscope using a Leica Aperio CS2 slide scanner (Wetzlar, Germany).

Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections using the Leica BOND RX (Leica, Wetzlar, Germany). Slides were first dewaxed and rehydrated. Heat-induced antigen retrieval was performed with citrate (pH 6) retrieval buffer for 20 min at 100°C. Primary antibodies were diluted 1:200 (H3K27me3) and 1:300 (H3K27acet, pJNK^T183/Y185) in Leica antibody diluent and incubated for 60 min on slides. Antibody staining was completed using the Bond Polymer Refine immunohistochemistry protocol and reagents (Leica, Wetzlar, Germany). Slides were counterstained on the Leica Autostainer XL (Leica, Wetzlar, Germany). Leica CV5030 glass coverslipper (Leica, Wetzlar, Germany) was used, and bright-field images were taken on the Aperio CS2 Slide Scanner (Leica, Wetzlar, Germany). Quantification of single-cell staining intensity was performed using the cell detection function of QuPath (v0.2.3).

Immunohistochemistry was performed on formalin fixed paraffin embedded sections using the Leica BOND RX (Leica, Wetzlar, Germany). Slides were first dewaxed and rehydrated, followed by heat induced antigen retrieval performed with Epitope Retrieval Solution 1 BOND (Leica, Wetzlar, Germany). Primary antibodies were diluted 1:600 (P70S6K) and 1:500 (Ki67) in Leica antibody diluent and incubated for 60 min on slides. Antibody staining was completed using the Bond Polymer Refine IHC protocol and reagents (Leica, Wetzlar, Germany). Slides were counterstained on the Leica Autostainer XL (Leica, Wetzlar, Germany). Leica CV5030 Glass Coverslipper (Leica, Wetzlar, Germany) and brightfield images were taken on the Aperio CS2 Slide Scanner (Leica, Wetzlar, Germany). Quantification of Ki67 staining was performed on three fields of view for each tumour section, and quantified using the particle analysis function of Image J (v1.49).

Immunohistochemistry was performed on formalin-fixed paraffin-embedded sections using the Leica BOND RX (Leica, Wetzlar, Germany). Slides were first dewaxed and rehydrated, followed by heat-induced antigen retrieval performed with Epitope Retrieval Solution 1 BOND (Leica). PCNA primary antibody was diluted 1:500 (Abcam, ab29) in Leica antibody diluent and incubated for 60 min on slides. Antibody staining was completed using the Bond Polymer Refine IHC protocol and reagents (Leica). Slides were counterstained on the Leica Autostainer XL (Leica). Leica CV5030 Glass Coverslipper (Leica) and brightfield images were taken on the Aperio CS2 Slide Scanner (Leica). Quantification of PCNA staining was performed on three fields of view for each tumour section using QuPath (v0.2.3)(Bankhead et al., 2017 (link)). Student’s t-test statistical analysis, along with dot plots and bar graphs, was generated using GraphPad Prism (v9.1.0).

Mice were lightly anesthetized with a mixture of O₂ and isoflurane and exposed once daily to either 80 μg of DEPs dissolved in 40 μL of PBS or PBS alone via the intranasal route for the duration of the experiment, unless otherwise stated. After 3 exposures, mice were anesthetized before intranasal infection with 1 × 10⁵ colony-forming units (CFU) of D39 in 10 μL of PBS. Control mice were treated with 10 μL of PBS only. Mice were humanely killed 1, 4, 7, or 10 days postinfection. The nasopharynx and lungs were removed from each mouse and blood was collected in heparin tubes. Bacterial viable counts were determined as described previously.⁴⁶ (link) Lung cell suspensions were stored at –80°C following erythrocyte lysis and supernatants were also stored at –80°C until required. For histologic analysis, lungs of PBS- or DEP-exposed mice were fixed overnight in neutral buffered formalin (10% formalin) followed by 95% ethanol and processed using standard histologic techniques. Six-micron frontal sections of the lungs were stained with hematoxylin and eosin and imaged using an Aperio CS2 Slide Scanner (Leica, Milton Keynes, UK). Apeiro Image Scope v12.3.2.8013 was used to analyze images.