Histomount

Four 4-μm sections were sequentially immersed in xylene, then immediately transferred to ethanol, and finally placed in a water bath for 15 min. Antigen retrieval was achieved by immersion of sections in preheated citrate buffer (0.25% trisodium citrate hydrate and 0.025% citric acid in deionized water, pH 6.1). The sections were then heated in a microwave oven (800 W for 10 min), cooled in citrate buffer, and rehydrated. Immunohistochemistry was carried out using the Novolink polymer detection system kit (Leica Microsystems, Newcastle, United Kingdom) according to the manufacturer's instructions. A monoclonal antibody (MAb) raised against the membrane-located l-α-glycerol-3-phosphate (GlpO) of M. mycoides subsp. mycoides (produced in-house at the International Livestock Research Institute [ILRI]), at the optimal dilution of 1:500, was used as primary antibody. The sections were dehydrated and visualized after mounting with Histomount (Vector).

Excised tissue was embedded in OCT (CellPath) and frozen using isopentane on dry ice. 8 µm sections were fixed in acetone and subjected to H&E staining or antibody labelling. For antibody labelling, sections were incubated sequentially with primary antibodies (Supplementary Table S1), followed by: Immunohistochemistry, ImmPRESS anti-rat or anti-rabbit IgG peroxidase polymer, Vector NovaRED peroxidase substrate, counterstained with haematoxylin and mounted using Histomount (all reagents, Vector Labs). Immunofluorescence, AlexaFluor488-conjugated anti-goat IgG and AlexaFluor568-conjugated anti-rat IgG (Life Technologies), counterstained with DAPI (Molecular Probes) and mounted with Vectashield (Vector Labs). Images were collected using a CKX41 inverted microscope reflected fluorescence system equipped with a CC12 color camera running under Cell B software (Olympus, UK). Composite immunofluorescence images were prepared using Adobe Photoshop (CS6).