Visualization of NHE and Cell Membrane

Immunofluorescence stainings for visualization of inflammasomes were described in our previous publication^{31 (link)}. For visualization of NHE and cell membrane, BMDMs were stimulated with NHE component C_WT or C_mut. or C + B for 1 h, or C + B + A for 30 min, washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min, followed by blocking in 1% BSA in PBS for 1 h. Cells were incubated with a rabbit sera anti-NHE-C (1:200 dilution in 1% BSA)^{55 (link)}, a mouse anti-NHE-B, or -A (1:200 dilution in 1% BSA)^{55 (link)}, an mouse anti-Histidine antibody (1:200 dilution in 1% BSA, ab18184, Abcam), and a rat fluorescein isothiocyanate-conjugated anti-CD11b antibody (1:200 dilution in 1% BSA, 101205, BioLegend) overnight at 4 °C. PBS containing 0.05% Tween-20 was used to wash between incubation steps. An anti-mouse secondary Rhodamine red antibody (115295146, Jackson ImmunoResearch) or rabbit secondary Rhodamine red antibody (111295144, Jackson ImmunoResearch) were used. The samples were mounted with VECTASHIELD Hardset Mounting Medium with DAPI (H-1500, Vector Laboratories, Inc.) and analyzed using a Leica SP5 confocal microscope.