Alexfluor 488 donkey anti rabbit igg

The following antibodies were used for immunoblotting and immunostaining: anti-IRX1 (Sigma HPA043160, 1:100 – 1:300), anti-IRX1 (abcam ab98343, 1:200 – 1:500), anti-Lef-1 (Cell signaling #2230, 1:200), anti-Sox2 (R & D systems AF2018, 1:150 & D systems AF2018, 1:150), anti-SP-C (Santa Cruz sc-7706, 1:300), anti-BrdU (abcam ab6326, 1:1000), anti-CIdU (Accurate chemical OBT0030, 1:250), anti-IdU (Roche 11170376001, 1:250). Secondary staining was performed using Alex Fluor488 donkey anti-mouse IgG (Invitrogen A21202, 1:500), Alex Fluor594 donkey anti-mouse IgG (Invitrogen A21203, 1:500), Alex Fluor488 donkey anti-rabbit IgG (Invitrogen A21206, 1:500) DAPI (Invitrogen D1306, 1 μg/ml), Beta-gal (Promega V3941).

FACS usually decreased cells vitality in our previous experiments. For acquiring cells with higher viability to perform three-dimensional organotypic cultures in vitro and epidermal reconstitution assay in vivo, we used magnetic cell sorting. Cells from newborn mice were incubated with primary antibodies (TLR7 antibody, sc-16245, Santa Cruz) binding to extracellular domain of TLR7 for 30 min, followed by incubation with biotinylated antibody at the appropriate concentration for 30 min. After washing with an excess volume of 1 × BD IMAG™ buffer, cells were incubated with BD IMAG™ streptavidin particles for 30 min. The labeling volume was increased to 2 × 10⁷ cells/ml with 1 × BD IMAG™ buffer, and the tube was transferred onto the BD IMagnet™ for magnetic cell sorting, following the manufacturer's guidelines. TLR7-positive cells were collected and subjected to flow cytometric analysis for the quantification of TLR7-positive cell enrichment. The positive fraction was labeled with TLR7 antibody (IMG-581A, Imgenex) binding to intracellular domain of TLR7 and AlexFluor 488 donkey anti-rabbit IgG (Invitrogen). After staining with 7AAD, the cells were analyzed by FACS (BD, FACS Aria).