Alexa fluor 594 conjugated goat anti mouse igg

Cells were seeded on to coverslips, irradiated with UVB light, and harvested at various time points. Afterward, 4% formaldehyde solution (15 minutes, room temperature) was act on fixed cells, the cell membranes were permeated with 1% Triton X-100 for 10 minutes, blocked with 5% goat or donkey serum for 60 minutes at 37°C, and cultivated overnight at 4°C with primary antibodies. The next morning, the cells were reacted at 37°C for 1 hour with the fluorescent secondary antibodies after washing 3 times. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology, China) for 5 minutes in the dark. All cell samples were imaged using laser confocal microscope (LSM 800; Carl Zeiss AG, Germany). The antibodies in this study: Hsp27 (1: 200; Cell Signaling Technology, USA) and p21 (1: 200; Abcam, UK), Alexa Fluor 594-conjugated goat anti-mouse IgG (1: 200; catalog no. SA00006-3; Proteintech, China), Dylight 488 goat anti-rabbit IgG (1: 200; catalog no. A23230-1; Abbkine Scientific, China), and Dylight 549 goat anti-rabbit IgG (1: 200; catalog no. A23320-1; Abbkine Scientific, China.).

Stably transfected HSC-3 cells were fixed with cold methanol for 5 min at −20°C, then washed with DPBS, and permeabilized with 0.25% Triton X-100 for 10 min. After blocking for 1 h with 1% BSA in DPBS/0.1% Tween-20, and rinsing with DPBS three times, the cells were incubated with rabbit anti-cathelicidin (cat. no. ab69484; Abcam) or mouse anti-LL37 (cat. no. sc-166770; Santa Cruz Biotechnology, Inc.) antibodies at room temperature for 1 h, followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 594-conjugated goat anti-mouse IgG (ProteinTech Group, Inc.) at room temperature for 1 h (27 (link),28 (link)). Nuclei were stained with 300 nM DAPI at room temperature for 1 min, and the cells were then imaged using an epifluorescence microscope (Nikon Eclipse Ti; Nikon Corporation; magnification, ×400).