Fluoroshield

Transfected cells were fixed with 4% paraformaldehyde for 20 min at RT, washed with PBS, and mounted with ProLong Antifade Mountant (Thermo Fisher Scientific, Rockford, IL, USA) for imaging. Samples were visualized using a Zeiss LSM 700 confocal microscope using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective. Zeiss ZEN 2010 program was used to control imaging specifications. The gap junction area was determined using ImageJ by tracing the gap junction area with a free hand tool followed by quantification using the measure tool. ImageJ software was also used to analyze co-localization data.
In the case of immunofluorescence, transfected cells were fixed with ice-cold 100% methanol for 10 min at RT. Cells were blocked using PBS supplemented with 2% BSA for 1 h at RT. Primary antibody, rabbit anti-HIS (Bethyl Laboratories Inc., Montgomery, TX, USA) at 1:1000 concentration, was diluted in PBS with 0.1% BSA and applied to cells for 1 h at RT. Cells were then incubated in the secondary antibody solution containing 2 μg/mL of Alexa Fluor 568 goat anti-mouse (Thermo Fisher Scientific, Rockford, IL, USA) and 2 μg/mL of Alexa Fluor 488 donkey anti-rabbit (Thermo Fisher Scientific, Rockford, IL, USA) in PBS with 0.1% BSA for 1 h at RT. Cells were washed in PBS and mounted with FluoroshieldTm (Sigma-Aldrich Chemie GmbH, Munich, Germany).

Briefly, coverslips containing human primary pulmonary alveolar epithelial cells (HPAEpiCs) were washed twice with phosphate-buffered saline (PBS, pH 7.6) and fixed with fresh high-grade 4% paraformaldehyde (PFA) for 10 min. The PFA was aspirated and the coverslips were washed 4 times in PBS for 5 min each time. The coverslips were blocked with 1.5% Normal Goat Serum (NGS, Sigma Aldrich UK) for 2 h at room temperature. Primary antibody incubation was carried out for 2 h at room temperature or overnight at 4 °C with gentle agitation. HPAEpiCs were identified by using a Zonula Occludens primary antibody (Rb polyclonal anti ZO-1 – Abcam ab96587). ZO-1 antibody was used in a 1:100 dilution and the samples were washed in PBS for 5 × 5 min. The corresponding secondary antibody (goat anti-rabbit IgG Abcam ab96883) conjugated to Dylight^® (1:1000) was added to the cells for 2 h at room temperature with gentle agitation. Cells were washed in PBS for 5 × 5 min, and the coverslips were inverted onto a drop of mounting medium containing DAPI (Fluoroshield^TM Sigma Aldrich UK) on a microscope slide, and stored at 4 °C. The immunostained cells were viewed under fluorescence (Nikon Eclipse 80i fluorescent microscope) with the appropriate excitations for each fluorophore. All images were analysed using ImageJ-Win64.

LnCAP or RWPE-1 cells were seeded in glass chamber slides and siRNA was performed as described above. After five days, cells were fixated with 1.6% paraformaldehyde, blocking was performed using 5% goat serum (Abcam, Cambridge, UK) and 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in TBS. Cells were incubated with primary antibodies targeting β-tubulin (9F3; Cell Signalling Technology, Danvers, MA, USA) or nuclear pore complex proteins (Mab414, Abcam) o/n at 4 °C. Incubation with the secondary antibodies (Alexa Fluor^® 488 Goat anti-rabbit, Abcam; Alexa Fluor^® 546 Goat anti-mouse, Invitrogen) was performed for 1 h at RT. Before mounting the samples with Fluoroshield^TM (Sigma-Aldrich), DAPI staining was performed for 3 min at RT. Images were taken using a NIKON C2 Eclipse Ti microscope (Nikon, Tokyo, Japan).

Dissociated rat DRG primary cells were seeded at 5000 cells/well in 6-well plates. After 48 h, the cells were taken from the culture, washed three times with PBST, and fixed in 4% paraformaldehyde for 15 min. Before blocking with 3% BSA for 2 h, cells were permeabilized with 0.5% Triton-X 100. DRG primary cells were treated overnight with anti-rabbit p-AMPK (Thr 172) in 3% BSA. The next day, cells were washed with PBST and treated with anti-mouse rhodamine for 2 h at room temperature. Coverslips were placed using DAPI mounting media (FluoroshieldTM, Sigma, St. Louis, MO, USA). Confocal images were taken (Leica TCS SP8 Laser Scanning Spectral Confocal microscope, Wetzlar, Germany) [23 (link),37 (link)].

Mid-modiolar cochlear cryosections were washed with 0.1 PBS (3 × 10 min), permeabilized with 1% Triton X-100 and blocked with 10% normal goat serum (NGS) in 0.1 M PBS for 1 h at room temperature. Mid-modiolar cochlear cryosections were incubated overnight at 4 °C with the rat anti-mouse TLR-2 monoclonal antibody (CD282; Invitrogen, Carlsbad, CA, USA, Product# 14-9021-82; 2.5 μg/mL) in 0.1% Triton X-100 and 10% NGS in 0.1M PBS. The following day, the sections were incubated in Alexa Fluor 488 goat anti-rat IgG (Invitrogen, Carlsbad, CA, USA, Product# A-11006; 4 μg/mL) for 2 h at room temperature. The cryosections were then washed three times with 0.1M PBS (2 × 10 min and the final 40-min wash), then mounted on microscope glass slides using FluoroshieldTM with DAPI mounting medium (Sigma-Aldrich).

AOs were fixed with 4% paraformaldehyde for 24 h at RT, followed by dehydration using sucrose solution of a gradual gradient series. The fixed AOs were then embedded in Tissue-Tek^® cryomold (Sakura finetek) covered with Tissue-Tek^® OCT^TM Compound (Sakura finetek) rocking at 4 °C overnight and stored in −80 °C. Frozen AO blocks were sectioned into 8 μm cryosections, which were carefully mounted onto silane-coated micro slides (Muto Pure Chemicals) and stored in −80 °C. The sections were blocked by 10% donkey serum (Jackson ImmunoResearch Laboratories) in PBST for 1 h at RT and probed with primary antibodies (EPCAM, CPM, HOPX, SFTPC, AQP5, T1α and VIMENTIN) in 1% donkey serum in PBST at 4 °C overnight. The next day, the sections were rinsed with PBST for 5 min, followed by incubation with secondary antibodies for 30 min at RT. The sections were counterstained using Fluoroshield^TM with DAPI histology mounting medium (Sigma-Aldrich). IHC images were captured under a fluorescence microscopy (IX-51, Olympus). Primary antibodies were omitted in control immunohistochemical staining. The antibodies are listed in Supplementary Table 3.

Procedures for immunostaining, fluorescence microscopy and NET quantification are described in the Supplementary Materials file. Briefly, neutrophils were stimulated with 5 µM ionomycin ((IO), Abcam, Cambridge, MA, USA) for 2–2.5 h at 37 °C in a humidified atmosphere with 5% CO₂, washed and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). The cells were immuno-stained overnight at 4 °C with antibodies of interest, followed by incubation with secondary antibody fluor staining. The neutrophils were next counterstained and mounted with Fluoroshield^TM (Sigma Aldrich), which contains 4′,6-Diamidino-2-phenylindole (DAPI) (Sigma Aldrich). Images were acquired on a fluorescence microscope. Morphologic quantification of NETs was performed on the basis of strict morphological criteria by two investigators as described in the Supplementary Materials file.