K550 sputter coater

Mid-logarithmic phase bacteria were washed and resuspended in PBS before peptide treatment at 100 µg/mL for 12 h or protein treatment at 1.84 µM for 6 h on coverslips. Treated bacteria were washed with PBS and post-fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 3 h and further washed with PBS. Cells were stained in 1% osmium tetroxide in distilled water for 1 h and dehydrated in a series of alcohol. Dehydrated samples were dried using liquid carbon dioxide critical point drying. Samples were then sputter-coated with platinum (3 nm thickness) at 15 mA for 2 min using the EMI TECH K550 Sputter coater and visualized under a Zeiss UltraPlus Field emission scanning electron microscope at 5 kV.

Bone marrow‐derived macrophages or mid‐logarithmic phase cultures of M. catarrhalis incubated either in the presence or absence of recombinant mGBP2 were washed in PBS, fixed with 2.5% glutaraldehyde in PBS for 3 h and further washed with PBS. Cells were fixed in 1% osmium tetroxide in distilled water for 1 h. Samples were subsequently dehydrated in a series of alcohol and subjected to liquid carbon dioxide critical point drying. Samples were then sputter‐coated with platinum (3 nm thickness) at 15 mA for 2 min using the EMI TECH K550 Sputter coater and visualised under a Zeiss UltraPlus Field emission scanning electron microscope at 2–5 kV.

Lecithinase‐ or ATP‐treated BMDMs were washed with PBS, fixed with 2.5% glutaraldehyde in PBS overnight and further washed with PBS. Cells were fixed in 1% osmium tetroxide in double distilled water for 1 h and gradually dehydrated in a series of ethanol. Dehydrated samples were transferred to a critical point dryer to replace ethanol by liquid carbon dioxide and dried completely. Samples were then sputter‐coated with platinum (3 nm thickness) at 15 mA for 2 min using the EMI TECH K550 Sputter coater and visualized under a Zeiss UltraPlus Field emission scanning electron microscope in secondary electron mode at 5 kV.

BMDMs were washed with PBS and post-fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer overnight and further washed with PBS. Cells were fixed in 1% osmium tetroxide in double distilled water for 1 h and dehydrated in a series of alcohol. Dehydrated samples were dried using liquid carbon dioxide using critical point drying. Samples were then sputter-coated with platinum (3 nm thickness) at 15 mA for 2 min using the EMI TECH K550 Sputter coater and visualized under a Zeiss UltraPlus Field emission scanning electron microscope at 5 kV.