12. Transfer the homogenate to a 2 mL screw-cap tube, and use a
further 500 μL PBS-Tw (0.05%) to rinse out the homogenizer
bag, adding this rinse to the same tube (total ¼ 1 mL homogenate in tube).
13. Plate serial dilutions of the homogenate on 7H10 agar for
determination of bacterial load.
14. Place the remaining 3 right lung lobes in a 15 mL conical tube
containing 3 mL of 4% PFA, and fix overnight at room temperature, protected from light.
15. The next day, surface-decontaminate the tubes and remove
them from the BSL-3 facility.
16. Transfer the fixed lungs to tubes containing PBS and store at
4
C (see Note 12).
3.5 Analysis of M.
tuberculosis-Host
Interactions In Vivo,
with Retention of
Spatial Information,
Using Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. To embed a fixed lung lobe, pour a small volume of 4% w/v agarose in PBS into a 5 mL disposable polystyrene sample beaker
(just sufficient to cover the base of the beaker).
2. After ~1 min (to allow the base layer to slightly gel), lay the
lung lobe on top, and overlay with additional 4% agarose in
PBS to fully embed the sample. Use forceps to gently recenter
the lobe as needed.
3. Wrap the sample carefully in plastic wrap and allow it to gel
completely at 4
C, protected from light (>1 h).
4. Carry out all storage and incubation steps described below with
the samples wrapped in foil to protect from light exposure.
5. Trim the agarose block containing the lung lobe to a rectangular block with the sample in the middle, and secure it to the
specimen disc of the vibratome with cyanoacrylate adhesive.
6. Cut 250 μm sections (0.145 mm/s, 70 Hz, blade angle 5
),
and place them into cold PBS (see Note 13).
7. Use forceps to carefully tease away any agarose that does not
come off the section automatically. Store all sections in PBS at
4
C.
8. Stain the sectioned samples in 1.5 mL microcentrifuge tubes.
Place the desired section in a 1.5 mL microcentrifuge tube
containing 500 μL of blocking buffer and incubate it on a
nutator for 1 h at room temperature.
9. Prepare primary antibodies in blocking buffer, and centrifuge
the working solution at 13,000 rpm (16,000 Â g) for 5 min
immediately before use (only the supernatant is used) (see
Note 14).
10. Move the sample ( from step 8) to a new 1.5 mL microcentrifuge tube containing 200 μL of primary antibody solution and
incubate overnight on a nutator at room temperature.
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David Giacalone et al.
further 500 μL PBS-Tw (0.05%) to rinse out the homogenizer
bag, adding this rinse to the same tube (total ¼ 1 mL homogenate in tube).
13. Plate serial dilutions of the homogenate on 7H10 agar for
determination of bacterial load.
14. Place the remaining 3 right lung lobes in a 15 mL conical tube
containing 3 mL of 4% PFA, and fix overnight at room temperature, protected from light.
15. The next day, surface-decontaminate the tubes and remove
them from the BSL-3 facility.
16. Transfer the fixed lungs to tubes containing PBS and store at
4
C (see Note 12).
3.5 Analysis of M.
tuberculosis-Host
Interactions In Vivo,
with Retention of
Spatial Information,
Using Reporter M.
tuberculosis-Infected
Murine Lung Tissue
1. To embed a fixed lung lobe, pour a small volume of 4% w/v agarose in PBS into a 5 mL disposable polystyrene sample beaker
(just sufficient to cover the base of the beaker).
2. After ~1 min (to allow the base layer to slightly gel), lay the
lung lobe on top, and overlay with additional 4% agarose in
PBS to fully embed the sample. Use forceps to gently recenter
the lobe as needed.
3. Wrap the sample carefully in plastic wrap and allow it to gel
completely at 4
C, protected from light (>1 h).
4. Carry out all storage and incubation steps described below with
the samples wrapped in foil to protect from light exposure.
5. Trim the agarose block containing the lung lobe to a rectangular block with the sample in the middle, and secure it to the
specimen disc of the vibratome with cyanoacrylate adhesive.
6. Cut 250 μm sections (0.145 mm/s, 70 Hz, blade angle 5
),
and place them into cold PBS (see Note 13).
7. Use forceps to carefully tease away any agarose that does not
come off the section automatically. Store all sections in PBS at
4
C.
8. Stain the sectioned samples in 1.5 mL microcentrifuge tubes.
Place the desired section in a 1.5 mL microcentrifuge tube
containing 500 μL of blocking buffer and incubate it on a
nutator for 1 h at room temperature.
9. Prepare primary antibodies in blocking buffer, and centrifuge
the working solution at 13,000 rpm (16,000 Â g) for 5 min
immediately before use (only the supernatant is used) (see
Note 14).
10. Move the sample ( from step 8) to a new 1.5 mL microcentrifuge tube containing 200 μL of primary antibody solution and
incubate overnight on a nutator at room temperature.
374
David Giacalone et al.