Alex fluor 488 anti mouse

UAS-Beat-Vb x Tub-GAL4 or wild-type embryos (Figure 5) were collected, dissected, and stained following procedures described in Lee et al (Lee et al., 2009 (link)). Similar methods were used for wild-type embryos in Figure 5—figure supplement 2. Dissected embryos were stained with Side-VI-AP (in S2 media), followed by primary antibodies rabbit anti-AP (Serotec) and mAb 1D4. Secondary antibodies used were Alexa-Fluor 568 anti-rabbit and Alex-Fluor 488 anti-mouse (Invitrogen) at a 1:1000 dilution. Images were collected on a Zeiss LSM 710 using a 40X objective.

RAW 264.7 cells were seeded on a Nunc™ Lab-Tek™ II Chamber Slide™ System (Nunc; Thermo Fisher Scientific, Rochester, NY, USA) and incubated for 2 h in a CO₂ incubator to allow the cells to adhere to the bottom of the slide well. Then, cells were applied to a differentiation medium and 300 µM of melatonin. The medium was replaced daily for 5 days. Cells were fixed in 4% formaldehyde solution (Sigma-Aldrich, Louis, MO, USA) for 10 min, and 0.2% Triton X-100 was applied for 10 min at room temperature to allow the antibody to penetrate the cells. Subsequently, to block nonspecific binding, the cells were blocked with PBS including 1% bovine serum albumin (BSA) for 1 h. The cells were incubated in the primary antibody (diluted at 1:100; NFATc1 and TRAF6 (Cell Signaling Technology, Beverly, MA, USA)) at 4 °C overnight. The cells were washed 3 times with PBS, then the secondary antibody (diluted at 1:200; Alex Fluor 488 anti-mouse; Invitrogen Gaithersburg, MD, USA) was incubated for 1 h at room temperature. Then, rhodamine phalloidin (Invitrogen, Eugene, OR, USA) was applied for 30 min to stain actin, and DAPI (Invitrogen, Eugene, OR, USA) was applied for 10 min to stain the nucleus. The expression of each protein was scanned and analyzed using a Zeiss LSM 750 laser scanning confocal microscope (Göttingen, Germany).