that the recovery of Leptospira in Subheading 3.5 is from
intracellular.
10. Cold methanol postfixation can be skipped if cathepsin D is not
stained.
11. After this step, the procedure can be stopped, and the coverslips can be stored in TBS at 4
C.
12. Exchange the solutions quickly, and avoid drying of the coverslips during all the immunostaining procedure.
13. The dilution ratio and the incubation time may vary depending
on the antibody (we used 1:50 dilution in TBS for
cathepsin D).
14. After the addition of the first fluorochrome-conjugated secondary antibody, perform all incubations and washing steps in
the dark to prevent fluorochromes from fading.
Fig. 1 Acquisition of LAMP-1 and cathepsin D by the Leptospira-containing phagosomes. BMDMs were
infected with either live or heat-killed L. interrogans at a MOI of 50 as described in Subheading 3.3. At the
indicated time points, cells were fixed and processed for immunofluorescence as described in Subheading
3.4. DIC: differential interference contrast. Scale bars, 5 μm
Macrophage Escape Assays
205
intracellular.
10. Cold methanol postfixation can be skipped if cathepsin D is not
stained.
11. After this step, the procedure can be stopped, and the coverslips can be stored in TBS at 4
C.
12. Exchange the solutions quickly, and avoid drying of the coverslips during all the immunostaining procedure.
13. The dilution ratio and the incubation time may vary depending
on the antibody (we used 1:50 dilution in TBS for
cathepsin D).
14. After the addition of the first fluorochrome-conjugated secondary antibody, perform all incubations and washing steps in
the dark to prevent fluorochromes from fading.
Fig. 1 Acquisition of LAMP-1 and cathepsin D by the Leptospira-containing phagosomes. BMDMs were
infected with either live or heat-killed L. interrogans at a MOI of 50 as described in Subheading 3.3. At the
indicated time points, cells were fixed and processed for immunofluorescence as described in Subheading
3.4. DIC: differential interference contrast. Scale bars, 5 μm
Macrophage Escape Assays
205