Vectashield mounting medium for fluorescence with dapi

To determine the effect of honokiol on the formation of NADPH complex, melanoma cells (Hs294t and SK-Mel 28) were treated with various concentrations of honokiol (0, 10 and 20 μM) for 24 h. The cells were then harvested and used for cytostaining of NADPH complex proteins, such as p47^phox and p22^phox. Briefly, the cells were cultured on coverslips in 100 mm culture plates with or without treatment with honokiol. After 24 h of treatment, cells were fixed with chilled 4% paraformaldehyde and non-specific binding sites were blocked with 2% bovine serum albumin in PBS for 30 min. Cells were then incubated with primary antibodies specific to p47^phox or p22^phox for 2 h at room temperature. The cells were washed with PBS and bound antibodies were detected using an AlexaFluor-conjugated secondary antibody. Goat anti-mouse IgG labeled with green-fluorescent AlexaFluor488 dye was used for detection of p22^phox, while goat anti-mouse IgG labeled with red-fluorescent AlexaFluor594 was used for the detection of the expression of p47^phox. Cells were finally mounted with Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories, Burlingame, CA). Immunofluorescence detection was performed using a fluorescence microscope and representative images were collected.

Muscle cryosections were blocked with 5% goat serum/1% BSA in PBS and incubated with antibodies listed in Supplementary Table 2. After washing with PBS, specimens were incubated with secondary antibodies labelled with Alexa Fluor 488 or Alexa Fluor 568 (Invitrogen, 1:1,000) and mounted in VECTASHIELD Mounting Medium for Fluorescence with DAPI (Vector Laboratories). Images of the immunofluorescent staining were recorded using fluorescence microscope Biozero BZ-8000 system (KEYENCE). Fibre size was quantified using Image J software (National Institutes of Health).

Placental explants of 5 µm sections were prepared on adhesive glass slides (Superfrost Plus™, Thermo Fisher Scientific) deparaffinized, counterstained with DAPI (Vectashield^® Mounting Medium for Fluorescence with DAPI, Vector Laboratories, Szabo-Scandic GmbH, Vienna, Austria) and analyzed, using a fluorescence microscopy system Leica TCS SP2 (Leica Microsystems GmbH, Vienna, Austria) equipped with a digital camera (AxioCam HRc, Carl Zeiss GmbH, Vienna, Austria).
Biotinylated dPG-NPs were detected, using a Streptavidin-Peroxidase-AEC detection system (Thermo Fisher Scientific).
After exposure to dPG-NPs, BeWo cells from coverslips and primary trophoblast cells from chamber slides were washed three times using HBSS (Gibco^®), fixed with 4% PFA for 20 min and counterstained with DAPI for evaluation on the confocal microscope. Confocal microscopy was performed to distinguish possible NP deposition on the outer cell surface from intracellular dPG-NP deposits by choosing the optical sections at the estimated equatorial level of the nuclei for analysis. Amira software version 3.1 (TGS Template Graphics Software Inc., Bedford, UK) was used for 3D reconstructions from Z-stacks.

Immunofluorescence analysis was performed as previously described [21 (link)]. The slides were microwaved in AR9 buffer (Opal-4 Color Manual IHC kit; PerkinElmer, Waltham, MA, USA) and cooled for 30 min. Sections were then incubated in Antibody Diluent/Blocking Buffer (Opal-4 Color Manual IHC kit; PerkinElmer) for 10 min at room temperature and then incubated with primary antibodies (listed above). Triple staining was performed with antibodies against IL-8 and SerpinE1 overnight at 4 °C and antibodies against CD206 for 2 h at room temperature. Samples were washed three times in TBST for 2 min each and then incubated in Polymer HRP (Ms + Rb) (Opal-4 Color Manual IHC kit; PerkinElmer). Samples were rinsed and then washed three times in TBST for 2 min each and then incubated in Opal Fluorophore working solution (TSA Plus System; PerkinElmer) for 10 min at room temperature. Samples were rinsed in TBST, microwaved in AR9 buffer, and then mounted with VECTASHIELD Mounting Medium For Fluorescence with DAPI (Vector Laboratories, Burlingame, CA, USA). Photomicrographs were obtained using a light microscope with a digital camera (BZ-X800 series; Keyence, Tokyo, Japan). The ratios of triple IL-8-, SerpinE1-, and CD206-positive cells in immune cells and tumor areas were quantified using the BZ-H4C and BZ-H4CM analytic applications.