Prolong dapi mounting medium

For 24-h microculture, 600 μL of 20 × 10⁴ cells/mL suspension of cells was instilled into each well of Millicell EZ 8-well glass slides (Merck). Microcultures with cells were incubated at 37 °C for 24 h. Then, the cells were fixed using 4% formaldehyde. Subsequently, the fixed cells were incubated with the specific polyclonal rabbit anti-irisin/FNDC5 antibody (dilution 1:50; code no. NBP2-14024; Novus Biologicals) at 4 °C overnight. The slides with the fixed cells were rinsed and incubated for 1 h with donkey anti-rabbit secondary AlexaFluor 568 conjugated antibody (dilution 1:2000; code no. A10042; Invitrogen, Waltham, MA, USA). The secondary antibody was diluted in a background-reducing reagent (Agilent Technologies, Santa Clara, CA, USA). The Prolong DAPI Mounting Medium (Invitrogen) was used to stain the cell nucleus and mount the slides. The observations were made at ×600 magnification using Olympus Fluoview FV3000 confocal microscopy coupled with CellSense (Olympus) software.

Antibodies and dilutions used are summarized in Supplementary Methods. Cells were fixed in 4% paraformaldehyde (PFA), blocked with blocking buffer (PBS with 10% fetal bovine serum, FBS; 30 min, room temperature), permeabilized (5 min, room temperature) with 0.1% Triton-X100, and incubated with primary antibodies (overnight, 4 °C). After washing, cells were incubated with an appropriate secondary antibody (1 h, room temperature); washed cells were mounted in Prolong DAPI mounting medium (Invitrogen) and viewed under a fluorescent or confocal microscope.

Immunofluorescence protocols have been previously described in detail. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100 (5 min, room temperature), blocked with blocking buffer (PBS with 5% BSA; 30 min, room temperature) and then incubated with primary antibodies in PBS/1% BSA (overnight, 4 °C). Slides were washed three times in PBS/1% bovine serum albumin (BSA) and incubated in PBS/1% BSA with appropriate secondary antibodies (1 h, room temperature). Washed cells (three times in PBS/1% BSA) were mounted in Prolong DAPI mounting medium (Invitrogen). Images were captured with a Zeiss LSM 700 or a Leica TCS SP5 confocal microscope. Supplementary Methods Table 2 section online includes the list of all primary and secondary antibodies used. Fluorescence intensity was measured using ImageJ software (NIH, Bethesda, MD) (https://imagej.net/software/imagej/).

To perform IF, 24-h microculture cells were placed on slides (Millicell EZ 8-well glass slides; Merck). For microculture, 600 μL of 20 × 10⁴ cells/mL suspension was instilled into each well on the slides. Microcultures with cells were placed in an incubator at 37 °C for 24 h. After the incubation, the cells were fixed with the use of 4% formaldehyde and further incubation was conducted with the specific polyclonal rabbit anti-irisin/FNDC5 antibody (dilution 1:50; code no. NBP2-14024; Novus Biologicals) at 4 °C overnight. After rinsing, the slides were incubated for 1 h with polyclonal donkey anti-rabbit secondary AlexaFluor 568 conjugated antibody (dilution 1:2000; code no. A10042; Invitrogen, Carlsbad, CA, USA) in the reagent with a background-reducing component (Agilent Technologies). The slides were mounted using the Prolong DAPI Mounting Medium (Invitrogen). The observations were made at x600 magnification with the use of Fluoview FV3000 confocal microscopy (Olympus) coupled with CellSense software (Olympus, RRID:SCR_016238).

For immunofluorescence (IF), deparaffinisation, and hydration, thermal epitope demasking was done using a low pH Target Retrieval Solution (Agilent Technologies) for 20 min at 97°C in a Dako PT Link (Dako) apparatus. Sites of non-specific binding were blocked using 3% BSA in PBS (1 h/RT). The slices were incubated at 4°C overnight with primary anti-POSTN antibodies (dilution 1:200; code no. NBP1-82472; Novus Biologicals) in 3% BSA/PBS, anti-Podoplanin (ready-to-use, clone D2-40 (PDPN), code ISO072; Dako), and anti-αSMA (ready-to-use, clone IS611, code 1A4; Dako). Next, the preparations were incubated for 1 h with donkey anti-rabbit secondary Alexa Fluor 568 conjugated antibody (dilution 1:2000, Abcam, Cambridge, UK, Cat#ab175470, RRID:AB_2783823) and anti-mouse Alexa Fluor 488 conjugated antibody (dilution 1:2000, Abcam, Cambridge, UK, Cat# ab150113, RRID:AB_2756499).
Negative controls were performed with 1% BSA in PBS instead of the specific antibody. The preparations were mounted in a Prolong DAPI Mounting Medium (Invitrogen, Carlsbad, CA, USA). The observations were made at objective 60×/1.40 oil; with the use of a Fluoview FV3000 confocal microscopy (Olympus, RRID:SCR_017015) coupled with Cell Sense software (Olympus, RRID:SCR_016238).

Cells were fixed in 4% PFA, blocked with blocking buffer (PBS with 10% FBS; 30 min, room temperature), permeabilized with 0.1% Triton-X100 (5 min, room temperature), and then incubated with primary antibodies (overnight, 4 °C). After washing, cells were incubated with an appropriate secondary antibody (1 h, room temperature), washed cells were mounted in Prolong DAPI mounting medium (Invitrogen), and analyzed under a fluorescent or confocal microscope. Antibodies are summarized in Supplementary Table S6.

For microculture, 600 µL of 2 × 10⁴ cells/mL suspension of cells was set up on slides with Millicell EZ 8-well glass slides (Merck,) and placed in an incubator at 37 °C for 24 h. After the incubation, the cells were fixed using 4% formaldehyde. The slices were incubated at 4 °C overnight with primary specific polyclonal rabbit anti-NUCB2 (1:1000 dilution; code no. NBP2-35072; Novus Biologicals) at 4 °C overnight. Next, the preparations were incubated for 1 h with donkey anti-rabbit secondary Alexa Fluor 568 conjugated antibody (1:2000 dilution; clone, code no.; Invitrogen, Carlsbad, CA, USA) and were mounted using the Prolong DAPI Mounting Medium (Invitrogen, United StatesI co). The observations were made at objective 60×/1.40 oil using Fluoview FV3000 confocal microscopy (Olympus, RRID:SCR_017015) coupled with Cell Sense software (Olympus, RRID:SCR_016238).