Immunofluorescence

Cells were cultured for 24 h on sterilized coverslips and then for a further 24 h after treatment with either complete medium, 100 ng/mL LPS and 20 ng/mL IFN-γ, 20 ng/mL IL-4, 500 μM phenelzine, or 500 μM GSK to form the treatment groups: control, M1 (IFN-γ + LPS), M2 (IL-4), phenelzine, and GSK, respectively.

After culturing, cells were fixed with 3.7% paraformaldehyde and permeabilized using 2% Triton X-100 solution. Cells were then blocked using 1% bovine serum albumin (BSA) and probed with rabbit-LSD1p (ABE1462, EMD Millipore, Burlington, MA), mouse-H3K9me2 (ab1220, Abcam, Cambridge, UK), goat-H3K4me2 (ab11946, Abcam), mouse-CD38 (102716, BioLegend), and goat-SNAIL1 (sc-10433, Santa Cruz Biotechnology) followed by visualization with corresponding secondary antibodies (all Thermo Fisher Scientific): anti-rabbit (A21206 and A10042), anti-mouse (A10037), and anti-goat (A21082 and A11055) conjugated to either Alexa Fluor 488, or 568, or 633. Coverslips were mounted onto glass microscope slides using SlowFade™ Diamond Antifade Mountant with DAPI.

Formalin-fixed, paraffin-embedded melanoma primary tumor biopsies were processed in the BondRX for OPAL staining (Perkin-Elmer, Waltham, MA) using the instrument protocol: ER1 for 20 min at 100°C with Epitope Retrieval Solution (pH6 Citrate-based retrieval solution) followed by probing with primary antibodies to F4/80 (ab100790, Abcam), iNOS (ab115819, Abcam), CD86 (ab213044, Abcam) and PD-L1 (ab2386097, Abcam) (for the M1 panel) or F4/80, EGR2 (ab90518, Abcam), CD206 (ab64693, Abcam) and PD-L2 ({"type":"entrez-protein","attrs":{"text":"PAB12986","term_id":"1236625678","term_text":"PAB12986"}}PAB12986, Abnova) (for the M2 panel). Primary antibodies were visualized with an Opal Kit 520, 570, 650, and 690. Coverslips were mounted on glass microscope slides with ProLong Clear Antifade reagent (Life Technologies, Carlsbad, CA). Opal kits used: 7-color automation kit (NEL801001KT) and the 4-color automation kit (NEL820001KT).

Slides were observed under a Leica DMi8 inverted microscope running Leica Application Suite X software. Multiple images were taken at various positions on the slide using a 100x oil immersion lens. Images were analyzed using ImageJ software, with the fluorescence intensity measured from a minimum of 20 cells and an average total fluorescence of either the nucleus or cytoplasm reported. Background fluorescence was measured and subtracted from all results.

For high-throughput microscopy, protein targets were localized by confocal laser scanning microscopy. Single 0.5 μm sections were obtained using an Olympus-ASI automated microscope with 100x oil immersion lens running ASI software. The final image was obtained by employing a high throughput automated stage with ASI spectral capture software. Digital images were analyzed using automated ASI software (Applied Spectral Imaging, Carlsbad, CA) to automatically determine the distribution and intensities with automatic thresholding and background correction of either the average nuclear fluorescent intensity (NFI) and average or whole cell total fluorescent intensity (TFI). The plot-profile feature of ImageJ was used to plot the fluorescence signal intensity along a single line spanning the nucleus (n = 5 lines per nucleus, 5 individual cells) using the average fluorescent signal intensity for the indicated pair of antibodies plotted for each point on the line with SE. Signal was plotted to compare how the signals for each antibody varied compared to the opposite antibody. For each plot-profile, the PCC was determined in ImageJ. PCC indicates the strength of relationship between the two fluorochrome signals for at least 20 individual cells ± SE. Colors from representative images correspond to plot-profiles.