Anti fade mounting medium

To further confirm hNIS and 3flag expression in MSCs transduced with the LV_EF1a-3flagFluc-T2A-hNIS-IRES-PuroR and the LV_EF1a-3flagFluc-IRES-PuroR, immunofluorescent stainings were performed. Cells were fixed using unifix for 20 min at 4°C, permeabilized with 0.05% Triton for 30 min at room temperature (only in case of 3flag staining) and blocked with 10% normal donkey serum (Millipore) for 20 min at room temperature. Cells were then incubated for two hours at room temperature with the primary antibody diluted in PBS (hNIS 1/20 and flag 1/1000). Fluorescently labeled secondary antibody (1∶500, Alexa Fluor donkey anti-rabbit 488 or donkey anti-mouse 555, Invitrogen) was incubated for 30 min at room temperature. Nuclei were counterstained using DAPI and sections were mounted using anti-fade mounting medium (Dako). Images were acquired using a Nikon Eclipse 80 i Fluorescence microscope equipped with a Nikon DS-2 MB Wc digital camera (Nikon Tokyo, Japan). Stainings in which primary antibodies were omitted, were used as a negative control.

The sections were air-dried, fixed in ice-cold 4% paraformaldehyde (in PBS) and washed in PBS. Non-specific binding was blocked with 10% normal goat serum in PBS for 30 minutes. For immunofluorescence analysis of myosin heavy chain (MHC), sections were first incubated with specific primary monoclonal antibodies (BA-F8 for MHCI, SC-71 for MHCIIa and BF-F3 for MHCIIb, all from Developmental Studies Hybridoma Bank, University of Iowa, USA) for 2 hours at room temperature, washed, followed by incubation with iso-type specific Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Thermo Fisher Scientific, USA) for 1 hour at room temperature in dark. Sections were mounted with antifade mounting medium (DAKO) and visualized under fluorescence microscope (40X objective, Eclipse 80i; Nikon Instrument Inc, Japan). Images were captured using Spot Advanced software (Diagnostic Instruments Inc, USA). Individual images were taken across the entire cross-section and assembled into a composite panoramic image with Microsoft Image Composite Editor (Microsoft). All fibers within the entire muscle/cross-section were characterized for fiber type analyses. We then computed the percentages of each type of muscle fiber.

Cells seeded on glass coverslips were fixed in 4% PFA, and immunostainings were performed according to a standardized protocol [4 (link), 7 (link)]. In summary, in the case of an intracellular epitope, cells were permeabilized with 0.05% Triton X-100 in PBS at 4°C for 30 minutes. Afterwards, 10% donkey serum was used to block nonspecific binding sites. Cells were incubated at room temperature for 1 hour with the primary antibodies listed in Table 1. Afterwards, cells were incubated with the appropriate secondary antibody at room temperature for 30 minutes. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and coverslips were mounted with antifade mounting medium (Dako, Glostrup, Denmark) on glass slides. Negative controls were included in each staining in which the staining procedure was performed in parallel with the other samples but with omission of the primary antibody. Micrographs were taken with a Nikon Eclipse 80i fluorescence microscope equipped with a 2MBWc digital sight camera and NIS-Elements software.

The colon was harvested (n = 4/group), cut along the mesenteric border and pinned to silicone-based petri dishes containing 1x phosphate buffered solution (PBS). Tissues were incubated in Zamboni’s fixative (2% formaldehyde, 0.2% picric acid and 0.1 M sodium phosphate buffer (pH 7.0)) overnight at 4°C. The following day, tissues were washed 3 x 10 minutes in 100% DMSO, followed by 3 x 10 minute washes with 1x PBS. Sections (30μm) were cut and incubated with a mouse blocking reagent (M.O.M. kit, Vector Labs, USA), or 10% normal donkey serum for 1 hour at room temperature, then washed 3 x 10 minutes with PBS and Triton-x100 (PBS-T). Sections were co-labelled with anti-TLR4 (mouse, 1:500, Abcam, USA) and anti-HMGB1 (rabbit, c-terminus, 1:500, Abcam, USA) antibodies or with anti-CD45 (mouse, 1:500, Abcam, USA) antibody alone. Primary antibodies were incubated at room temperature overnight, and were then washed 3 x 10 minutes with PBS-T. The secondary antibody for both TLR4 and CD45 was FITC-conjugated (mouse, 1:200, Abcam, USA) made up in M.O.M. diluents; and the secondary antibody for HMGB1 was AlexaFluor-647-conjugated (rabbit, 1:200, Jackson Immuno-Research, USA). Secondary antibodies were incubated at room temperature for 2 hours and then washed 3 x 10 minutes with PBS-T. Sections were mounted onto glass slides using an anti-fade mounting medium (DAKO, Australia).

Cells were fixed with 2.5% formalin in phosphate-buffered saline (PBS) for 10 min. Fixed samples were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, blocked for 1 hour with Protein Block (Dako), and incubated with 2 μg/ml rabbit polyconoclonal anti-human TLR3 (#LS-B4866, Sigma, Aldrich, USA), overnight at 4°C. In negative controls, the primary antibody was replaced with PBS. Excess primary antibody was removed, and 2 μg/ml anti-Rabbit CY3 conjugated secondary antibody (Jackson ImmunoResearch Labs Inc., West Grove, PA, USA) was added and incubated for 1 hour at RT. The Membranes were rinsed in TBST, and after the third wash, 200 ng/ml of 4′, 6-diamidino-2-phenylindole (DAPI; Sigma, Aldrich, USA) was added to resolve nuclei. Membranes were transferred to a glass slide and a drop of anti-fade mounting medium (Dako, Glostrup, Denmark) was added before cover-slipping. Samples were visualized by using a LSM700 confocal laser scanning microscope (Zeiss Microscopy, Germany). Slide tissue was prepared as above except tissue were cut in 4 µm sections from CRSwNP patients, deparaffinized and rehydrated. Antigen retrieval was induced at 100 °C for 10 minutes in 10 mmol/L sodium citrate buffer, pH 6.

Aliquots of 1 × 10⁵ cells from PBMCs were cytospun for 5 minutes at 500 rpm using a centrifuge(Thermo Shandon, Pittsburgh, PA, USA). The slides were air dried and fixed in methanol for 10 minutes. Then, the cells were blocked with 1% BSA/PBS at room temperature for 30 min and incubated with antibodies specific for H3K9me3(GeneTex, San Antonio, TX, USA) or SUV39H1(Novus Biologicals, Littleton, CO, USA) at room temperature for 40 min. After washing, the cells were incubated with an anti-rabbit antibody Alexa Fluor^® 488(Abcam, Carlsbad, CA, USA) at room temperature for 1 hr. After washing, the cells were incubated with 4′,6-diamidino-2-phenylindole dihydrochloride(DAPI) at room temperature for 10 min. Immunostaining cells were mounted on slides with anti-fade mounting medium(DAKO, Japan). Lung tissues were obtained from COPD patients(patients undergoing lung surgery for peripheral lung tumor removal). Lung function testing was performed before lung cancer surgery. Normal control tissues were derived from noninvolved lung segments of the tumor lesion. IHC was performed on paraffin-embedded human or mouse lung tissue sections using rabbit antibodies against SUV39H1 and H3K9me3, as previously described29 (link). Images were acquired at 200x magnification and analyzed by Nikon NIS Elements D Imaging Software Analysis(Nikon, Tokyo, Japan).