Preparation of lung tissue for histological and immunohistochemistry analysis
Lungs of mice were perfused with absolute ethanol by endotracheal route and fixed for 24 h and then embedded in paraffin blocks. Sections of 4μm width were obtained, mounted on glass slides, deparaffinized and stained with hematoxylin and eosin. For quantification and morphometric analysis determining the surface area affected by pneumonia, three different mouse lungs per time point were evaluated in two independent experiments.
For immunohistochemistry, lung tissue was sectioned and mounted on charged glass slides and then deparaffinized. Slides were first blocked for unspecific activity of peroxidase with methanol peroxide 3% for 1 h. For 11-βHSD1and 11-βHSD2 tissue detection, rabbit anti-mouse monoclonal antibodies (Biorbyt) were used at a concentration of 1:250, incubated overnight in agitation, followed by incubation with secondary anti-rabbit IgG labeled with peroxidase. For cortisone and corticosterone immunostaining, rabbit antimouse monoclonal antibodies (LsBio) were used at a final concentration of 1:250. In both cases, bound antibodies were detected with diaminobenzidine and counterstained with hematoxylin.
Immunohistochemistry stained sections were analyzed by digital pathology. Briefly, stained sections were digitized at 200 x magnification using an Aperio ScanScope CS (Aperio). The Aperio ScanScope CS images had a spatial resolution of 0.45 μm/pixels. The images were analyzed using an ImageScope (Aperio). All the lung tissue sections were circumscribed and sent for automated image analysis using the Spectrum Software V11.1.2.752 (Aperio). For immunostaining GCs intensity quantification, an algorithm was developed to determine the total lung expression of cortisone and corticosterone in the whole tissue section (26–28).