Manual sputter coater

Mycobacteria (5 × 10⁵ CFU/mL in diluted RPMI medium) were transferred into a 24-well-plate containing poly-l-lysine-coated round cover glasses. After addition of IL-26 or LL37, the mycobacteria were incubated for 24 h. The mycobacteria were fixed with 2.5% glutaraldehyde and 4% paraformaldehyde (PFA). After refrigerated overnight incubation, the cover glasses were first washed with PBS followed by a serial dehydration starting with 50% ethanol (EtOH) and increasing to pure EtOH. Thereafter the cover glasses were washed twice with 100% acetone. The dehydrated samples were then subjected to critical point drying (CPD). The dried cover glasses were sputtered with gold using a Manual Sputter Coater (Agar Scientific, Essex, UK). Scanning electron microscopy (SEM) was performed using a Leo 1430 VP (Zeiss, Jena, Germany).

The images of the insect and the microstructure of the elytra were captured using a Lynx LM-1322 optical microscope (Olympus corporation, Tokyo, Japan) and a charged coupled device (CCD) camera (Nikon, Tokyo, Japan) attached to the microscope. Scanning Electron Microscope (SEM) imaging of all the samples is performed after cleaning with the help of ultrasonication and a subsequent drying. The prepared elytra sections were carefully mounted on double-sided carbon tape and stuck on an aluminum stub, followed by sputter coating (Manual sputter coater, Agar Scientific Ltd., Stansted, United Kingdom) with gold. Images were captured using an SEM (EVO 40 XVP, ZEISS, Jena, Germany) with accelerating voltages between 5 and 20 kV. ImageJ software was used for all dimensional quantification reported in this study [26 ].

The SaOs2 cell morphology was studied by SEM. For SEM studies, the cells were fixed after 1 and 3 days of interaction with samples. For fixation, 2.5% glutaraldehyde was used for 45 min at room temperature and washed two times with PBS. Samples were kept in PBS until the process of dehydration. This procedure involved successive immersion in 70%, 90%, and 100% ethanol (EtOH), 15 min twice for each concentration. Cells were then incubated by sequential incubation in 50%:50%, 25%:75%, and 0%:100% solutions of EtOH:hexamethyldisilazane (HMDS), two times for 3 min in each combination. The specimens were dried and metalized prior to the microscopy investigations. The metallization consisted of a 10 nm thin gold layer deposited by magnetron sputtering on the surface of specimens with a manual sputter coater (Agar Scientific, Essex, UK).