Dmire2

MELC was employed for multiplex IF-staining of the herein established 20-plex antibody panel, as described [36 (link)].
Briefly, MELC is based on repetitive cycles of antibody staining and photobleaching. After system start, four fields of view are selected and calibration (brightfield and darkframe) images are acquired. Prior to every staining and photobleaching cycle with the acquisition of the corresponding fluorescence tag and post-bleaching image, the slide is washed with PBS and a phase-contrast image is taken.
Camera (ApogeeKX4, Apogee Instruments) and light source maintain the same position; the motor-controlled xy stage of the inverted fluorescence microscope (Leica DMIRE2, Leica Microsystems; x20 air lens; numerical aperture, 0.7) moves in between fields of view. Images with a resolution of 2018 × 2018 pixels are acquired, with one pixel corresponding to 0.45 µm at a 20× magnification. Thus the whole image covers a field of view covering 908.1 × 908.1 µm.
Additionally, negative control secondary antibodies were implemented, which were applied to the sample prior to indirect staining of the respective primary antibody.
The subsequently applied interphase fluorescence in situ hybridization (iFISH) is described in the supplementary methods.

Mice were dissected to collect abdominal subcutaneous fat (abdominal subWAT), epididymal WAT (eWAT), retroperitoneal WAT (rWAT), intestinal WAT, and the liver. Immediately after the collection, tissues were weighed and processed for further analysis. For adipocyte staining, the eWAT and liver were fixed with 10% neutral formalin and embedded in paraffin. All the tissue samples were sectioned at 6 μm and stained with hematoxylin and eosin (H & E). Images were captured using an inverted confocal microscope (Leica DMIRE2; Leica Microsystems, Wetzlar, Germany) with a 63× oil immersion objective lens. All the images were captured with the same laser intensities.

The eWAT (epididymal WAT) was fixed and washed with phosphate-buffered saline (PBS). The sections were exposed to a primary antibody against UCP-1 (sc-518024, Santa Cruz Biotechnologies Inc., CA, USA) for 2 h at room temperature. Following incubation with a primary antibody, the sections were washed thrice for 5 min in PBS. Later, the sections were labeled using anti-mouse IgG- FITC (Sigma, St Louis, MO, USA) for 1 h at room temperature. The sections were washed thrice for 5 min in PBS, and glass coverslips were mounted with 20 μL aqueous-mount solution (Scytek laboratories, Logan, UT, USA). Images were captured using an inverted confocal microscope (Leica DMIRE2; Leica Microsystems, Wetzlar, Germany) with a 63× oil immersion objective lens. All the images were captured with the same laser intensities.

Isolated LDs were analyzed and quantified using a fluorescence microscope. In addition, direct samples were taken from the cell cultures to determine and quantify LDs within cells (LDs/cell). For that purpose, the lipophilic TopFluor^® Cholesterol or Bodipy Cholesterol dye [23-(dipyrrometheneboron difluoride)-24-norcholesterol] (Avanti^® Polar Lipids, AL, United States) was used for selective labeling of LDs. The dye was first dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL. Then, as in Zhou et al. (2019) (link), 2 or 3 drops (300 mL approx.) of TopFluor^® Cholesterol were added to 1 mL of culture medium to obtain a final concentration of 10 mM. The solution was stirred and left for 10 min in the dark at room temperature. Finally, the supernatant was removed and washed with distilled water before observation.
The freshly stained LDs were observed under a fluorescence microscope using a 10X ocular and 10X objective lens (Leica DMIRE2, Leica Microsystems Inc., Wetzlar, Germany) coupled to a camera (Leica DFC360-FX) with red and green fluorescence filters (excitation, 545 and 480 nm; emission, 620 and 535 nm, respectively), exposure of 1.233 s and gain 1.55. Isolated LDs and LDs/cell were quantified using the Java-based image processing program imageJ and were expressed as number of LDs or LDs/cell per area (LDs/cm² and LDs/cell/cm², respectively).

Differential expression analysis was performed on multiplex imaging-based single-cell data³⁵ of monocytes and macrophages derived from neuroblastoma bone marrow samples. Briefly, multi-epitope ligand cartography is based on repetitive cycles of antibody staining and photobleaching. After system start, four fields of view are selected and calibration (brightfield and darkframe) images are acquired. Prior to every staining and photo-bleaching cycle with the acquisition of the corresponding fluorescence tag and post-bleaching image, the slide is washed with PBS and a phase-contrast image is taken. Camera (ApogeeKX4, Apogee Instruments) and light source maintain the same position; the motor-controlled xy stage of the inverted fluorescence microscope (Leica DMIRE2, Leica Microsystems; x20 air lens; numerical aperture, 0.7) moves in between fields of view. Images with a resolution of 2018 × 2018 pixels are acquired, with one pixel corresponding to 0.45 µm at a 20× magnification. Protein expression, represented by the mean of the 20% highest pixel intensities was compared between cells derived from samples without and with tumor infiltration using the Wilcoxon–Mann–Whitney test with FDR correction. Data analysis and visualization was performed in python v3.9.12⁷⁴ using statannot v0.2.3 (https://github.com/webermarcolivier/statannot) and seaborn v0.11.2 packages^{75 (link)}, respectively.

Fresh weight was determined by filtering the cells with 80 mm Nylon filters. The cells were freeze-dried to obtain the dry weight and perform the taxane extraction. Cell viability was evaluated as described by Exposito et al. (2010) (link). A small aliquot of each sample was incubated for 5 min in B5 medium containing 0.01% (w/v) propidium iodide (ICN Biomedicals, Costa Mesa, CA, United States) for the selective labeling of dead cells, and 0.01% (w/v) fluorescein diacetate (Sigma Aldrich, St Louis, MO, United States) for the selective labeling of live cells. Fluorescence was observed under a fluorescence microscope (Leica DMIRE2, Leica Microsystems Inc., Wetzlar, Germany) coupled to a camera (Leica DFC360-FX) and using specific filters. Samples were harvested after 0, 8, 12, 16, 20, and 24 days of treatments.