Fluoroshieldtm

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.

UAS-rigor mortis KK RNAi lines (VDRC 105403) were crossed with ubiquitous inducible driver, Tubulin Gene switch (TubGS)-Gal4. Day 1 adults from the F1 progeny were collected every 24 h and moved to standard media mixed with 20 mM RU486 at 28 °C. Locomotion was assessed using the RING assay^{53 (link),54} . Briefly, flies were transferred, without anesthetization, into plastic vials and placed in the RING apparatus. The vials were tapped down against the bench and the climbing was recorded on video at day 20. Quantifications were performed manually by a third party in a blinded manner.
For studying NMJs defects, 3rd instar larvae expressing rig mortis RNAi were dissected, and fixed by using 4% formaldehyde. The RNAi was expressed using TubGS-Gal4 by growing the 1st instar larvae on 1 mM RU486 at 28 °C until they reach the 3rd instar stage. The larvae (n = 4) were probed with mouse anti-horseradish peroxidase (HRP), a presynaptic neuronal marker to identify the NMJs, for overnight at 4 °C. On the next day, the larvae were washed with 0.1% TBST and stained with goat anti-mouse Alexa fluor-568 secondary antibody. The larvae were mounted with fluoroshield^TM (Sigma) and images were taken at ×60 using Nikon A1-T216.3 confocal microscope.