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).