Biology Reference
In-Depth Information
10. Incubate coverslips on 50 μL-drops of Alexa Fluor™ 488-conjugated anti-mouse
secondary and Cyanin 5-conjugated anti-rabbit secondary antibody dilutions
(1:500) for 40 min at room temperature. This step allows the detection of
cytoplasmic bacteria and calnexin at the cytoplasmic face of the ER that have
been recognized following digitonin permeabilization.
11. Wash each coverslip twice in 0.1% saponin-PBS and once in PBS.
12. Incubate coverslips a second time with a mouse anti-
Francisella
LPS antibody
dilution for 40 min at room temperature in permeabilization/blocking buffer.
Because cells are fully permeabilized with saponin, this step allows the antibody
to reach all intracellular bacteria.
13. Wash each coverslip twice in 0.1% saponin-PBS and once in PBS.
14. Incubate coverslips with an Alexa Fluor™ 568-conjugated anti-mouse secondary
antibody dilutions or for 40 min at room temperature in permeabi-
lization/blocking buffer to detect all intracellular bacteria.
15. Wash each coverslip twice in 0.1% saponin-PBS, once in PBS, and finally once
in water to remove salts. Drain all liquid onto a Kimwipe and mount coverslips
on microscopy slides by inverting them (cells facing down) on 10-μL drops of
Mowiol mounting medium. Allow at least2hofpolymerization before viewing
samples on a confocal fluorescence microscope equipped with 488-, 568-, and
643-nm laser lines.
16. Under such staining conditions, permeabilized cells are detectable with the
643-nm (or equivalent) laser line. Cytoplasmic bacteria are detectable using both
488- and 568-nm (or equivalent) laser lines while phagosomal bacteria are only
detected using the 568 nm (or equivalent) laser line. Alternate combinations of
secondary antibodies are possible.
4. Notes
1. All solutions should be prepared in bi-distilled sterile water, which is referred
to as “water” in the text.
2. Mowiol is extremely slow to dissolve and may require overnight stirring before
heating up to approcimately 50 °C to complete its dissolution.
3. Once thawed, aliquots can be kept at 4 °C for about 15 days. It is best to prepare
large batches from which many experiments can be performed using the same
conditioned medium. The preparation of L-929-conditioned medium is crucial
for the differentiation of bone marrow monocytes into macrophages. Batches
should be tested for their ability to differentiate monocytes into macrophages
and maintain macrophages with proper spindle-like morphology for about 10
days following differentiation.
4. The cultures must be homogeneous (i.e., only macrophages) and cells must be
more or less spindle shaped. The presence of cells with heterogenous shapes
indicates that the original bone marrow isolate contained too many already mature
macrophages that produced M-CSF or other such molecules involved in differen-
tiation of precursor cells into dendritic cells, granulocytes, and so on. If cells look
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