Tcs spe 2

To assess the uptake of Cur-NPs in vitro, MDCK cells were treated for 30 min with five different inhibitors of endocytic pathways (EIPA, LY, CPZ, HS and MβCD).34 (link) These cells were then treated for 1 h with C6-NPs, after which the supernatant was removed and cells were washed thrice with PBS. Cells were then fixed for 5 min with 4% paraformaldehyde (PFA) and imaged via confocal laser scanning microscopy (CLSM; TCS SPE II, Leica, Germany).

Back skin or oral tissues (tongue) from p45 animals were fixed in 4% Paraformaldehyde for 0.5–1 hr followed by PBS washes. Tissues were incubated in 15% and 30% sucrose gradient at 4°C prior to embedding in O.C.T compound (Tissue Tek, cat. #: 4583) without prior fixation. Tissues were sectioned to 8µm on a Leica CM1950 cryostat using DB80LX blades (cat. #: 14035843496), mounted on SuperFrost Plus glass slides (Fisher Scientific, cat. #: 12–550-15), and dried 10 min at 37°C. Fresh frozen tissue from p0 mice and fixed p45 skin sections were fixed for 5 min with 4% PFA, washed with PBS, and blocked in a gelatin block (5% normal donkey serum (Jackson Immunoresearch, cat. #: 017–000-121), 3% BSA, 8% gelatin (Sigma cat. #: G7765), and 0.05% Triton X-100 in PBS) for 1 hr. Sections were stained with primary antibodies diluted in gelatin block and incubated overnight at 4°C, washed three times with PBS, incubated 2 hr with secondary antibodies at room temperature, counterstained with DAPI for 5 min at room temperature, and mounted in ProLong Gold (Invitrogen, cat. #: P36930). Tissues were stained with primary and secondary antibodies (Table S6) and images acquired using LAS AF software on a Leica TCS SPE-II 4 laser confocal system on a DM5500 microscope with ACS Apochromat 20 3 /0.60 multi-immersion, ACS Apochromat 40 3 /1.15 oil, and ACS Apochromat 63 3 /1.30 oil objectives.

Untreated and HU-treated (at a concentration of 2.5 mmol/L for 24 hours) organoids, grown on chamber slides (Falcon Culture-Slides) previously precoated with BME, were fixed in 4% paraformaldehyde in PBS solution for 30 minutes at room temperature and permeabilized with 0.5% Triton-X100 in PBS for 30 minutes at room temperature. Organoids were then incubated with 1% BSA in PBS for 60 minutes, followed by incubation overnight with the following primary antibodies diluted in PBS containing 1% of BSA and 1% of donkey serum: anti-RAD51 (Millipore ABE257; 1:100), anti-phospho-RPA32 (Ser33; Bethyl Laboratories A300-246A; 1:500), and anti-phospho-Histone H2AX (Ser139; Bethyl Laboratories A300-081A; 1:600). After washing, organoids were fluorescently labeled with Alexa Fluor 488 donkey anti-rabbit antibody (Invitrogen) diluted 1:400 in PBS containing 1% BSA and 1% donkey serum for 1 to 2 hours. Nuclei were stained with DAPI. Slides were then mounted using the fluorescence mounting medium (Dako) and analyzed using a confocal laser scanning microscope (TCS SPE II, Leica).