Faramount mounting medium

Bacteria were made electro-competent using glycerol/mannitol density step centrifugation, as described in an established protocol (Warren, 2011 (link)). The plasmid pBC20 with a gene encoding for the fluorescent protein Ypet (517/530nm) downstream of the constitutively active PybaJ promoter was electroporated into E. coli and K. pneumoniae. To visualize possible cell invasion of bacteria, A549 cells were seeded onto sterilized coverslips inside 6-well plates and infected with fluorescent bacteria at a multiplicity of infection (MOI) of 10 for 2h. Subsequently, cells on coverslips were washed thrice with PBS (Lonza) and fixed with 4% paraformaldehyde solution for 20min. Samples were permeabilized with 0.2% Triton X-100 (Roth) for 30min. Alexa-flour-594-labeled phalloidin (Invitrogen, A12381) was used to stain the actin cytoskeleton, and 4′,6-diamidino-2-phenylindole (DAPI, BioLegend, 422801) was used to stain nuclei for 30 min at room temperature. Samples were mounted with Faramount Mounting Medium (Dako, S3025) onto slides. Imaging was performed immediately after sample preparation using a VS120-S6 fluorescence microscope (Olympus). Images were captured with a 40-x objective using 387/440nm (DAPI), 485/525nm (Ypet), and 560/607nm (phalloidin) lasers and filters.

Lipid content was measured in HL-1 cells challenged with HP/HI in the presence or absence of AICAR. Cells were fixed in ice-cold 4% paraformaldehyde for 15 min and stained with fresh Oil-Red-O (Sigma) solution for 30 min. Nuclei were counterstained with Haematoxylin and cells were mounted with Faramount mounting medium (Dako) after extensive washing. Pictures were taken at 40x magnification with a Nikon digital camera DMX1200 and ACT-1 v2.63 software from Nikon Corporation. The lipid content was quantified by Image J software from five random fields of three different experiments.

For monitoring autophagosome formation or autophagy flux, GBM cells on glass coverslips were transiently transfected with GFP‐LC3 plasmid, followed by treatment with 10 μm PD for 24 h or were infected with lentivirus expressing DsRed‐LC3‐GFP, followed by treatment with 10 μm PD or 1 μm rapamycin for 24 h, respectively. For staining of intracellular p62, GBM cells treated with 10 μm PD or 100 nm BafA1 for 24 h were permeabilized with 0.01% Triton X‐100 in PBS. After blocking in 2% BSA in PBS, the primary antibody against p62 (Santa Cruz, USA) was incubated overnight at 4 °C, followed by further incubation with Alexa594‐conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at RT. To stain acidic compartments, 50 nm LysoTracker Red DND‐99 (Thermo Fisher Scientific) was added to the medium for 30 min at 37 °C and 5% CO₂ prior to fixation. The nuclei were labeled with 4, 6‐diamidino‐2‐phenylindole (DAPI; Sigma, St. Louis, MO, USA). The coverslips were finally mounted using faramount mounting medium (Dako, Glostrup, Denmark). The images were examined under a Zeiss laser confocal microscopy (Carl Zeiss, Oberkochen, Germany) and analyzed in zen software (Carl Zeiss).

In brief, sinonasal tissues were dehydrated and embedded in the paraffin and sectioned at 3 µm diameters. After rehydration and blocking of the endogenous peroxidase activity with 3% H₂O₂/methanol, the sections were washed with Tris-buffered saline (TBS) and blocked (with PBS, pH 7.4, containing 2% bovine serum albumin (Sigma-Aldrich, Darmstadt, Germany), 0.1% Triton X-100, and 0.1% sodium azide) at room temperature (RT) to reduce nonspecific bindings for 30 min to hinder nonspecific binding [21 (link)]. Then, the sections were incubated with an appropriate concentration of the antibodies for 1 h at RT. The details of which are as follows: primary antibodies, including polyclonal rabbit anti-human IgA antibody (at 1:100 dilution; Abcam, Cambridge, MA, USA), anti-human IgA1 antibody (at 1:200 dilution; ab193187), anti-human IgA2 antibody (1:100; ab193169, Abcam), monoclonal mouse anti-human CD20 (at 1:200 dilution; clone L26, Dako, Glostrup, Denmark), anti-human CD138 (1:100; Clone M115) were applied. After 2 h incubation, the slides were washed with TBS for 10 min and again incubated for 45 min at 30 °C with EnVision™ (Dako). The samples were coun-terstained with Mayer’s hematoxylin stain and mounted in Faramount Mounting Medium (Dako), before microscopic examination.

Cells plated on glass coverslips were fixed in cold methanol at −20°C for 10 min or fixed in 1% formaldehyde in HBS at RT for 8 min. The fixed cells were treated with 0.25% Triton X‐100 in HBS at RT for 5 min and washed three times with HBS. After soaking in HBS containing 1% bovine serum albumin (BSA) at RT for 10 min, the samples were treated with primary antibodies at RT for 60–180 min, followed by washing three times with HBS and incubating with secondary antibodies at RT for 60 min (In some experiments, rhodamine‐phalloidin [#R415; Molecular Probes] was added to detect F‐actin). The samples were washed three times with HBS, shortly soaked in Milli‐Q water (Millipore), and mounted with Faramount Mounting Medium (#S3025; DAKO). Immunofluorescent micrographs were acquired using a fluorescence microscope (BX51 or BX53; Olympus), a fluorescence microscope with a disk scanning unit system (BX53‐DSU; Olympus), or a Spinning Disk Confocal Super Resolution Microscope (SD‐OSR; Olympus).

Before the immunocytochemistry assay, fixed cells isolated on the filters of the ScreenCell^® Cyto device were air-dried overnight at room temperature and then hydrated with tris-buffered saline (TBS; Dakocytomation, Glostrup, Denmark) containing 0.05% Tween 20. The antigens were retrieved with target retrieval solution pH 9 (ER1; Leica Biosystems, Wetzlar, Germany) at 95–99 °C for 20 min and rinsed with Bond Wash (Leica Biosystems). Isolated cells were treated for 5 min at room temperature with a peroxidase block solution (Leica Biosystems). Then, the samples were incubated for 30 min at room temperature with mouse anti-human Melan-A (Dako). A post-primary rabbit anti-mouse was then applied for 8 min followed by a polymer HRP anti-rabbit for an additional 8 min. A sequential incubation step with mouse anti-human CD45 antibody (Leica Biosystems) was applied for 30 min. Finally, a chromogenic staining using DAB-RED detection according to Leica Biosystems protocol and a counter-staining with Hematoxylin (DS9665, Leica Biosystems) for 10 min allowed the revelation of the antigen detection. After a final wash with distilled water, the ScreenCell^® Cyto filter was mounted on a glass slide with the Faramount mounting medium (Agilent Technologies, Santa Clara, CA, USA), and covered with an 8 mm diameter coverslip.