Whole lungs with trachea were removed, immersion fixed in 4% paraformaldehyde pH 7.0 (Carl Roth, Karlsruhe, Germany) for 48 hours and embedded in paraffin (Thermo Scientific, Waltham, MA). 4 µm thick whole-lung horizontal sections were stained with hematoxylin & eosin (Carl Roth, Karlsruhe, Germany) and scored in a blinded fashion by a veterinary pathologist for the following parameters: perivascular edema, pleuritis, necrosis, area of infection. For immunohistochemical detection of S. pneumoniae in paraffin embedded sections, antigen retrieval was performed with microwave heating (600 W) for 12 minutes in 750 ml 10 mM citric acid pH 6.0 (Merck, Darmstadt, Germany). Sections were incubated with a purified rabbit polyclonal antibody which recognizes epitopes in the capsule, the cell wall and the cytosol of S. pneumoniae (1:2000, kindly provided by Sven Hammerschmidt, Interfaculty Institute for Genetics and Functional Genome Research, University of Greifswald, Germany) at 4°C overnight and subsequently incubated with an alkaline phosphatase conjugated goat anti-rabbit secondary antibody (1:500, Vector, Burlingame, CA) for 30 minutes at room temperature as described in (18 (link)).
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