Anti mouse cy3

Hyper- and hypo-pigmented epidermal sheets were stained with a melanocyte marker, S100β by an en face staining technique adapted from a previously described method [23 (link)]. The protocol used a primary S100β antibody diluted in 3% milk (1:50; ab11178) (Abcam, Cambridge, MA) and a secondary antibody (anti-mouse-CY3 at 1:100; Abcam) with subsequent DAPI application [24 (link)]. During the course of the study, the production of one S100β antibody was discontinued by the company, and the antibody was replaced with a new one (1:100; ab52642), and stains were completed with anti-rabbit-CY3 secondary antibodies. For the pig model, n = 9 scars from n = 3 pigs were sampled to generate hyper- and hypo-pigmented epidermal sheets. Within each sheet, 5 high-powered fields (HPF) were imaged, and cells were counted at 40X magnification. Within each HPF, dendrites were counted in a representative cell in each picture to obtain an average count per HPF. For the patient samples, n = 8 scars from n = 8 patients were sampled to generate hyper- and hypo-pigmented epidermal sheets. Cells and dendrites were counted as described above.

Flies were collected at ZT2 (two hours after lights-on in LD 12:12) and ZT14 (two hours after lights-off in LD12:12), their heads were fixed in 4% paraformaldehyde and brains were isolated. After washing in 0.2% phosphate buffer saline with Triton X-100 (PBST) and 30 min of blocking in Normal Goat Serum (NGS) they were incubated overnight with anti-PDF primary antibody (PDF C7 1:500, Developmental Studies Hybridoma Bank). Next, samples were washed in PBST and incubated with secondary antibodies (1:500 anti-mouse Cy3, Abcam). Whole brains were mounted in Vectashield medium (Vector) and examined with a Zeiss Meta 510 Laser Scanning Microscope.

Immunofluorescence analysis was performed as described previously [8 (link), 12 (link)]. Briefly, FLAG-tagged KLHDC7B-transfected HeLa cells (3 × 10⁵ cells/12-well plate) on glass (Matsunami Glass Co. Ltd., Osaka, Japan) were incubated with 400 ng mL⁻¹ of SubAB or mt SubAB. Cells were fixed with 4% paraformaldehyde (PFA), rinsed three times with PBS, and incubated with the blocking buffer (5% goat serum and 0.3% Triton X-100 in PBS) at room temperature for 1 h. Cells were further incubated with anti-FLAG antibodies (#014-22383, WAKO) in 0.4% BSA/PBS buffer at 4 °C overnight, washed twice with PBS, and incubated with anti-mouse Cy3 (#ab97035, Abcam, Cambridge, UK) antibodies at room temperature for 1 h in the dark.
After the cells were washed with PBS, they were mounted on glass slides using Prolong Gold Antifade reagent with DAPI (ThermoFisher Scientific). FV10i-LIV confocal microscopy (Olympus, Tokyo, Japan) was used to visualize the stained cells and the images were arranged with Adobe Photoshop CS4.