After obtaining lung macrophages from humans and mice by bronchoalveolar lavage, or BAL, according to the text protocol, use trypan blue and a hemocytometer to perform a viable cell count on the BAL solution. Pipette 1 to 4 x 105 macrophages in 500 microliters of culture medium onto coverslips in the wells of 24-well plates. Then, incubate the cells at 37 degrees Celsius overnight for the cells to adhere.
Remove the culture medium and use PBS to wash the cells once. Then, add 2% paraformaldehyde/periodate/lysine or PLP fixative, and incubate the samples for 10 minutes. After using PBS to briefly wash the cells, add 0.2% Tween-20 in PBS, and incubate the cells for 20 minutes.
Next, to block the cells, add 10% chicken serum in 5% BSA diluted in PBS, and incubate the samples at room temperature for 30 minutes. Then, replace the block solution with a 1 to 100 concentration of primary and isotope control antibodies, and incubate the cells at room temperature for one hour. Following the incubation, use PBS to wash the cells before adding the corresponding secondary antibodies. Then, incubate the samples for 40 minutes at room temperature.
After washing the cells in PBS, mount the samples with DAPI-based mounting medium. Then, visualize the samples using a confocal microscope.
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