Vectashield hardset with dapi

A portion of the cardiac muscles were embedded in Tissue-Tek O.C.T. Compound (Sakura) and multiple thin sections (10 μm) were cut using a cryostat (Microm). Subsequently, the sections were stained with hematoxylin and eosin (H&E), Masson's Trichrome (Polysciences, Inc.) or using immunofluorescence techniques. Masson's Trichrome staining was carried out according to the manufacturer protocol with the addition of an one hour 10% formalin fix at room temperature (RT) prior to the fixation in Bouin's solution [50 ]. For immunostaining, the sections were fixed with 4% paraformaldehyde for 15 minutes at RT then blocked with 5% BSA/0.3% TritonX-100/PBS for one hour at RT, incubated with the primary antibody, 8-oxoG DNA Lesion (Santa Cruz), overnight at 4°C and incubated with secondary AlexFluor antibody (Invitrogen) at RT for 1 hour. Slides were mounted with Vectashield Hard Set with Dapi (Vector Laboratories). All images were taken with an Eclipse Ni microscope (Nikon).

Micrographs were obtained as previously described [56 (link)] with the following modifications. Cells were fixed in 4% paraformaldehyde for 10 min, then incubated in permeabilization buffer (PBS, 0.5% Triton X-100, 1% BSA) for 10 min. Primary and secondary antibodies were prepared at dilutions of 1:500 (CHIP-Sigma HPA043531) or (anti-c-myc, Sigma M4439) and 1:800 (Alexa-Fluor Goat anti-rabbit or Goat anti-mouse), respectively, in blocking buffer (PBS, 0.05% Triton X-100, 1% BSA). Coverslips were mounted using Vectashield Hardset with Dapi (Vector Laboratories). Cells were visualized using a Zeiss LSM 710 spectral confocal. Co-localization analysis was performed using the Coloc2 plugin (v 3.0.0) with Fiji [57 (link),58 (link)].

A549 cells (1.5 × 10⁵) were incubated in individual wells of a 24-well plate with coverslips placed in each well for 24 h. E. coli and Geitlerinema sp. LPS was initially diluted in PBS. Cells were treated with 100,000 ng/mL of E. coli LPS or 100,000 ng/mL of Geitlerinema LPS for 6 h. This concentration was used based on previous studies demonstrating a peak LPS response of immune cells in vitro [28 (link),29 (link)]. Cells were fixed with methanol at 4 °C overnight. Cells were washed three times with PBS-Tween (0.02%) and incubated in 5% normal goat serum for 1 h at room temperature to block nonspecific binding. After subsequent washing, cells were incubated at 4 °C overnight in primary antibodies to rabbit CD54/ICAM-1 (1:25 Cell Signaling, Danvers, MA, USA). Cells were washed of primary antibody, then incubated with anti-rabbit IgG (AlexaFluor 488, Cell Signaling #4412, Danvers, MA, USA) at room temperature for 1 h, washed again, and mounted in VectaShield HardSet™ with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained on a Nikon EclipseTi confocal microscope (Nikon Instruments, Melville, NY, USA). Image files were quantified by the MFI and mean gray value using imageJ software (National Institutes of Health (NIH), Bethesda, MD, USA). The experiment was performed independently three times.

HEK293 cells stably expressing mycKOR or mycMOR were grown on poly-D-lysine treated cover slips the day prior to the experiment. For D2DR experiments, HEK293 cells were plated on coverslips 48 h prior to the experiment and transiently transfected with HA-D2DR(L) 24 h prior. Cells were serum starved 5 h, then treated with MJ33 (10 µM), SP610025 (1 µM), naloxone (10 µM), or vehicle 30 min prior to 1 h treatment with norBNI (1 µM or 10 µM). CellROX Green (10 µM, Molecular Probes) was added during the last 30 min of treatment. Cells were rinsed in PBS and fixed 15 min with 4% PFA. Cells were mounted on glass slides with VectaShield HardSet with DAPI (Vector Laboratories), and imaged within 12 h. Cover slips were imaged on a Nikon upright fluorescent microscope with Nikon Elements AR v3.1 software (Nikon Instruments). To prevent photoactivation, exposure to light during sample generation and imaging was minimized. Two representative fields from each cover slip were imaged for CellROX (488 nm) and DAPI. Exposure times were held constant for every fluorophore throughout the entire experiment. Image intensities for between 7 and 20 cells per image were quantified using ImageJ v 1.42q (National Institute of Health), and these were averaged to give an average field intensity value. This was done for the two cover slip images and averaged to make one sample (n).