Bz 9000 fluorescent microscope

C2C12 cells were cultured on glass coverslips coated with L-Lysine (Nacalai Tesque). On day four of differentiation, myotubes after each treatment were fixed in 4% paraformaldehyde/phosphate buffer solution (PBS). C2C12 myotubes were permeabilized in 0.25% Triton X-100/PBS and blocked in 1% bovine serum albumin/PBS (blocking solution) for 1 h at room temperature. Myotubes were washed with PBS and incubated for 1 h in anti-muscle MHC antibody at a dilution of 1:250, washed with PBS, and incubated in Alexa Flour 488 conjugated goat anti-mouse antibody (Cell Signaling Technology, #4408) for 1 h, and washed with PBS. Antibodies were diluted in blocking solution. The samples were mounted in a 4’, 6-diamidino-2-phenylindole (DAPI)-containing mounting medium. Images were captured with a BZ-9000 fluorescent microscope (Keyence, Osaka, Japan). Representative images for each treatment were selected. The myotube width was measured by BZ II Analyzer software (Keyence). One hundred myotubes per treatment were measured from ten non-overlapping fields-of-view, and were used to evaluate myotube atrophy.

MoDCs were cultured on 8-well chambered glass slides (ThermoScientific, Pittsburgh, PA). After 20 min treatment with PA or PA+CLI-095, the MoDCs were fixed and stained with primary anti-human phospho NF-κBp65 monoclonal antibodies overnight at 4°C (Cell signaling Technologies, Danvers, MA) and secondary donkey anti-rabbit FITC (Biolegend). Slides were sealed after addition of mounting media with DAPI and photos were taken at 40x magnification on a Keyence BZ-9000 fluorescent microscope.

Cells were grown in an 8-well slide chamber (Thermo Fisher) until confluency. Cells were washed with HBSS and fixed in 2% paraformaldehyde. For antibody staining, cells were permeabilized in HBSS with 0.1% Triton-X, for a minimum of 30 min. Cells were incubated with the primary antibodies overnight at 4°C in a solution of 0.1% Triton-X and 1% BSA (v/v) in HBSS and subsequently washed and incubated with the secondary fluorescently labeled antibody (Alexa-555 goat anti-mouse, dil:1:1000; Thermo Fisher: A-21127) for 2 h at room temperature. After the final washing step, slides were mounted and imaged in a Keyence BZ-9000 fluorescent microscope. For F-actin staining, cells were permeabilized with 2% Triton-X in HBSS; preliminary results showed poor drug transport antibody staining with 2% Triton-X, while 0.1% Triton-X showed poor phalloidin-488 (Abcam: 176763) staining in both S9 and HNO97 cells. Antibodies: BCRP, dil:1:500 (Merck; MAB4155C3); Pgp, dil:1:1000 (Novusbio; NB600-1036); OCT1, dil 1:1000 (Novusbio; NBP1-59464).

Sections were blocked with 5% normal donkey serum (Vector Laboratories)/0.1%Triton-X/0.01 M PBS for 1 h and incubated with polyclonal goat anti-mouse aminopeptidase N/CD13 for pericyte coverage. After incubation with the primary antibody, sections were washed in PBS and incubated with Alexa fluor 647-conjugated donkey anti-goat. To visualize brain endothelial vascular profiles sections were incubated with Dylight 594-conjugated Lycopersicon esculentum lectin (Vector Labs, DL-1177; 1:200) for 1 h. For double staining of lectin and CD13, Dylight 594-lectin was incubated simultaneously with Alexa fluor 647-conjugated donkey anti-goat secondary antibody for CD13. All incubations were performed in dark to prevent fading of fluorescence. All images were taken with a BZ-9000 fluorescent microscope (Keyence Corp). Z-stack projections, pseudo-coloring and image analysis were performed using ImageJ software (US National Institutes of Health). Gain, digital offset, and laser intensity were kept standardized.

Cells grown in 2D or MPS were fixed with a 2% paraformaldehyde solution and subsequently permeabilized with a 0.1% Triton-X solution in Hank's Balanced Salt Solution (HBSS). Primary antibodies were incubated overnight at 4 °C in a solution of 0.1% Triton-X and 1% BSA (v/v) in HBSS. Secondary antibodies were incubated for two hours at room temperature the following day and samples were washed in the 0.1% Triton-X and 1% BSA (v/v) HBSS solution before imaging using a Keyence BZ-9000 fluorescent microscope (Keyence, Osaka, Japan). The antibody staining conditions used are described in Supplementary Information Table 1.