Intracellular Localization of Brucella abortus and Francisella tularensis in Primary Murine Macrophages - Bacterial Pathogenesis: Methods and Protocols - page 129

10. Mowiol mounting medium: add 2.4 g of Mowiol 4-88 (Calbiochem, San Diego,
CA)to6gofglycerol and stir to mix. Add 6 mL of water and leave stirring at
room temperature for several hours. Add 12 mL of 0.2 M Tris-HCl (pH 8.5) and
heat to approximately 50 °C until Mowiol dissolves. Clarify by centrifugation at
5000 ×g for 15 min, aliquot in airtight 1.5 mL microtubes, and store at -20 °C.
Once thawed, the Mowiol mounting medium is stable at room temperature for
several weeks ( see Note 2 ).
2.4. Phagosomal Integrity Assay
1. KHM buffer: 110 m M potassium acetate, 20 m M HEPES, and 2 m M MgCl2,
pH 7.3. Adjust pH using 1 M KOH and sterilize by 0.2 μm filtration. KHM buffer
can be stored at 4 °C for several weeks.
2. Digitonin solution: Prepare extemporaneously a 10 mg/mL solution of digitonin
(Sigma, D141) in sterile bi-distilled water. Heat to 95 °C for complete dissolution
and let cool to room temperature before use.
3. Mouse anti- Francisella LPS monoclonal antibody (U.S. Biological).
4. Rabbit polyclonal anti-calnexin CT (Stressgen Bioreagents), which recognizes the
cytoplasmic tail of the ER chaperone calnexin.
5. Reagents used for immunofluorescence staining ( see Subheading 2.3. ).
3. Methods
Intracellular localization of B. abortus or F. tularensis is performed on
infected murine bone marrow-derived macrophages at various stages of their
intracellular life cycle, using immunofluorescence staining of bacteria and
various intracellular organelles. Specific intracellular compartments are labeled
by the immunodetection of host proteins enriched or specifically present on
(or in) these compartments or by loading with specific fluorescent probes ( see
Table 1 ). The presence of bacteria in a given intracellular organelle is evaluated
by co-localization of bacteria- and organelle-specific fluorescent signals, either
by indirect epifluorescence or laser-scanning confocal fluorescence microscopy
( see Fig. 1 ). Following phagocytic uptake, both Brucella and Francisella are
located within an initial phagosome that interacts to various extents with the
endocytic pathway (4,9) . Maturation of the BCV into a replicative organelle can
be monitored through the subsequent acquisitions and exclusions of markers
of various organelles, namely early endocytic, late endocytic, and ER markers
(4,5,8,11) ( see Fig. 1 ). Francisella phagosomal escape into the macrophage
cytoplasm limits its interactions with membrane-bound compartments, yet early
interactions with the endocytic compartment are detectable and phagosomal
escape can be assessed using an assay based on the specific detection of