Alexa fluor 488 goat anti mouse immunoglobulin g igg

The following primary and secondary antibodies were used to stain cells and tissue sections for immunofluorescence in this study: Pax7 (1:50; Developmental Studies Hybridoma Bank), MyoD (1:200; C-20, Santa Cruz Biotechnology), myosin heavy chain (1:200; M4276, Sigma-Aldrich), desmin (1:200; H-76, Santa Cruz Biotechnology), Alexa Fluor 488–conjugated anti-GFP (1:250; A-21311, Thermo Fisher Scientific), Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG; 1:250; A-11029, Thermo Fisher Scientific), Alexa Fluor 488 goat anti-rabbit IgG (1:250; A-11034, Thermo Fisher Scientific), Alexa Fluor 555 goat anti-mouse IgG (1:250; A-21422, Thermo Fisher Scientific), and Alexa Fluor 555 goat anti-rabbit IgG (1:250; A-21424, Thermo Fisher Scientific). Alexa Fluor 555 phalloidin (1:50; Thermo Fisher Scientific) and Alexa Fluor 647 phalloidin (1:50; Thermo Fisher Scientific) were used to stain F-actin. Hoechst 33342 (1:1000; Thermo Fisher Scientific) and DRAQ5 (1:1000; Thermo Fisher Scientific) were used to counterstain cell nuclei.

The skin specimens were harvested at the end of the experiment, fixed with 10% formalin (Sigma-Aldrich, St. Louis, MO, USA), and embedded in paraffin for sectioning (4 μm thickness). The prepared samples were stained with hematoxylin (Sigma-Aldrich) and eosin Y (Samchun Chemical, Seoul, Korea) (H&E staining), TB staining (Sigma-Aldrich), and crystal violet (Sigma-Aldrich) and Gram’s iodine (Sigma-Aldrich) (Gram staining). The H&E-stained samples were used to measure epidermal thickness, and TB-stained samples were used to quantify the number of mast cells as previously reported (54 (link), 55 (link)). For immunohistochemical staining, the prepared sections were permeabilized with 0.2% (v/v) Triton X-100 (Wako, Osaka, Japan) and incubated with 4% (v/v) bovine serum albumin (Wako). Then, the samples were incubated with the following antibodies: mouse anti–keratin 14 (Abcam, Cambridge, CBE, UK), goat anti–MMP-9 (R&D Systems, Minneapolis, MN, USA), Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG; Thermo Fisher Scientific, Waltham, MA, USA), and Alexa Fluor 594 rabbit anti-goat IgG (Thermo Fisher Scientific). The samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (TCI America, Portland, OR, USA) and observed with a laser scanning confocal microscope (LSM 880; Zeiss, Jena, Germany) or a slide scanner (VS120-S5-W; Olympus, Tokyo, Japan).

After genome editing, fibroblasts were left in culture for 1 week. Cells were then plated in a 96-well clear imaging microplates (BD Falcon) at a concentration of 1 × 10^{4 (link)} cells/well in DMEM (0.4% FBS). After 24 h, cells were incubated in modified medium containing 0.4% of FBS, 16.7 μg/mL of ascorbic acid, 37.5 mg/mL of Ficoll 70, and 25 mg/mL of Ficoll 400 to generate macromolecular crowding conditions. Transforming growth factor beta (TGF-β; 1 ng/mL) was also added, and fibroblasts were left at 37°C for 72 h before being fixed in ice-cold methanol and permeabilized in 0.1% Triton-X-100 in PBS. Immunostaining was performed by overnight incubation at 4°C with anti-collagen type I antibody (C2456; Sigma–Aldrich) at 1:1,000 dilution in PBS. Cells were then incubated for 1 h at room temperature with secondary antibody Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG; A11001; Invitrogen) at 1:500 dilution and Hoeschst dye (H3570; Invitrogen) at 1:10,000 dilution in PBS. A further staining step with anti-αSMA antibody (C6198; Sigma–Aldrich) at 1:1,000 dilution in PBS was carried out for 1 h at room temperature. The culture plate was scanned using a CellInsight NXT HCS instrument (Thermo Fisher Scientific) at 10 × magnification.