Precision cover glasses

Cells were plated on coverslips (Marienfeld precision cover glasses 1.5 H) fixed in 4% paraformaldehyde for 10 min, and permeabilized with 0.5% TritonX-100 in PBS for 5 min. In the case of digitonin treatment to decrease cytosolic staining, cells were treated with PBS containing 0.01% digitonin for 5 min on ice, followed by fixation with 4% paraformaldehyde, as described previously^{45 (link)}. Fixed cells were incubated with primary antibodies against PLIN1 (20R-pp004; Fitzgerald Industries, 1:300), ATGL (2138S; Cell Signaling, 1:300), PMP70 (ab3421; Abcam, 1:300 (all fluorescence images of immunostained PMP70 except for Supplemetary Fig. 4d), SAB4200181; Sigma, 1:300 (fluorescence images of immunostained PMP70 for Supplementary Fig. 4d), PEX5 (ab125689; Abcam, 1:300 (fluorescence images of immunostained PMP70 for Fig. 6a), sc-23188; Santacruz, 1:200 (fluorescence images of immunostained PMP70 for Supplementary Fig. 4d)), Catalase (ab16771, Abcam, 1:300) overnight, washed three times for 5 min each, incubated in secondary antibody for 1–2 h, washed three times for 5 min each, and mounted on glass slides with mounting medium (Vectashield without DAPI; Vector Laboratories). Cells were observed and imaged using an LSM 700 confocal microscope, and an OMX SIM microscope.

Human bronchial epithelial cells (BEAS-2B cell line) were maintained and serially passaged in F-12 culture medium supplemented with 10% fetal calf serum (FCS), 1% penicillin and streptomycin, and 10 mM HEPES in 75-cm² culture flasks. For microscopy experiments, BEAS-2B cells were grown to confluency on coverslips (precision cover glasses thickness No. 1.5H; Marienfeld, Lauda-Königshofen, Germany). To test interaction of SapL1 lectin with epithelial cells, BEAS-2B cells were incubated with different concentrations of FITC-lectin (5 or 10 µg mL⁻¹ in F-12) for 1 h at 37 °C. To validate the specificity of this interaction, SapL1-FITC was co-incubated in presence of 2 mM methyl-α-L-fucopyranoside for 30 min at 37 °C and then added to the cells for 1 h. Supernatant was then removed, cells washed 3 times with F-12, once with PBS and then fixed with 4% paraformaldehyde for 15 min. After 3 washes with PBS, nuclei were stained with 4'-6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:1000 in PBS) for 5 min. Coverslip were mounted with Prolong Glass Antifade Mountant (Invitrogen) on Superfrost glass slides (Thermo Fisher Scientific). Images were acquired with an upright Olympus BX43 microscope.
Figures were created using PyMOL Version 1.8.4 (Schrödinger), ChemDraw Version 15, ESPript Version 3.0^{65 (link)} and PowerPoint 16.