Fluoremount g

Induced cells were fixed, washed as described above, and blocked with 2% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Then, the cells were stained with the following primary antibody in 2% BSA/PBS overnight at 4 °C: PE-conjugated anti-human CD146 (clone: P1H12) antibody (1:100; BD Biosciences; 550315). After two washes, the cells were stained with Hoechst 33258 (1:1000; FUJIFILM; 343-07961), 1 µg/mL Bodipy 493/503 (Invitrogen; D3922), and Alexa Fluor 555-conjugated anti-mouse IgG1 secondary Antibody (1:1000; Invitrogen; A-21127) for 1 h at room temperature. The stained cells were washed twice and mounted using Fluoremount g (Southern Biotech, Birmingham, AL, USA; 0100-01). The prepared samples were observed under an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany).

TARO cells transfected with pCheryy/P2A FLAG-Ly6H in cover glass were treated with or without 0.35 U/mL phosphatidylinositol-specific phospholipase C (PI-PLC; Thermo Fisher Scientific, Inc.) in OPTI-MEM (Thermo Fisher Scientific, Inc.) for 1 h at 37 °C, and fixed for 15 min with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer at pH 7.4, then rinsed three times with PBS. Thereafter, cells were blocked with the blocking buffer (5% normal donkey serum (NDS) in PBS) for 30 min. Primary antibodies (monoclonal anti-FLAG M2 antibody, 1:100 dilution;) were applied in the blocking buffer for 1 h. After two washes in PBS, incubation (1 h) with donkey Alexa Fluor 647-conjugated anti-mouse antibody in blocking buffer was performed. After washing with PBS, cells were incubated with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Merck Millipore) to visualise nuclei, washed again and post-fixed with 2% PFA in 0.1 M PB for 10 min. PFA was removed by three rinses with PBS, coverslips were mounted in Fluoremount-G (SouthernBiotech, Birmingham, AL, USA), and examined using an Olympus FV-1000 confocal system with a 60 × objective lens (N.A. = 1.35).

BALB/c mice were deeply anesthetized with sodium pentobarbital and then perfused through the aortic cone with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) at pH 7.4. Thereafter, skin from the shaved back of each mouse was removed and post-fixed in the same fixative overnight at 4°C, after which they were immersed in 20% sucrose in PB overnight at 4°C. The tissues were then frozen in OCT, cut into 15-μm sections on a cryostat, and thaw-mounted on MAS-coated slides (Matsunami Glass, Osaka, Japan). Immunofluorescent labeling was performed using our rabbit polyclonal anti-SLURP1 antibody (0.7 μg/ml; 26). Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200 dilution; Life Technologies) was used as the secondary antibody. Sections were then wet-mounted in Fluoremount-G (SouthernBiotech, Birmingham, AL, USA) and examined using an Olympus FV-1000 confocal microscope system (Tokyo, Japan) equipped with a 60x objective lens (N.A. = 1.35). Confocal images of skin sections were acquired using a single sectioning.