Begin with red fluorophore-labeled M. tuberculosis antigens immobilized on a polymer-coated coverslip.
Add a blocking buffer to block nonspecific binding sites. Wash to remove excess blocker.
Introduce total protein extracted from human macrophages, which includes the cell surface receptor of interest. Incubate.
The macrophage receptors bind to the immobilized bacterial antigens.
Add a cross-linker to stabilize the receptor–antigen complexes, followed by a quencher to neutralize the unreacted cross-linkers.
Wash and add a second cross-linker to reinforce the complexes.
Wash again. Then, remove the coverslip and invert it over a primary antibody solution.
The antibodies bind to the macrophage receptor of interest.
Wash and invert the coverslip over a green fluorophore-conjugated secondary antibody solution, which binds to the primary antibodies.
Wash and mount the coverslip.
Visualize the sample under a fluorescence microscope. Co-localized red and green signals confirm the interaction between the macrophage receptor and the bacterial antigen.
Using round-nosed surgical tweezers, place 12 millimeter round cover slips into the wells of a 24-well culture plate.
Incubate the cover slip with Mycobacterium tuberculosis rhodamine antigens for 1 hour at 37 degrees Celsius. After incubation, add 100 microliters of protein extract diluted in 300 microliters of PBS and incubate for 2 hours at room temperature with agitation. Wrap the lid of the culture plate with paraffin film.
Add a 60 microliter drop of previously titrated anti-SLAMF1 primary antibody onto the paraffin film-covered lid. Carefully remove the cover slip from the plate using curved fine-tip surgical tweezers and needles. Following incubation with primary and secondary antibody solution, remove excess liquid and mount the cover slip on a drop of mounting liquid placed on a glass slide. Place the slide under a fluorescence microscope and observe the cells using appropriate filters.
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