Anti irisin fndc5 antibody

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.

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).