Immunofluorescent Analysis of Smad2/3 in Gingival Fibroblasts

In line with our previous research^{21 (link)}, immunofluorescent analysis was conducted on human gingival fibroblasts plated onto Millicell EZ slides (Merck KGaA, Darmstadt, Germany) stimulated with ADL 10% for 30 min. Cells were fixed using paraformaldehyde and blocked in 5% BSA and 0.3% Triton in PBS at room temperature for 1 h. Cells were next incubated with Smad2/3 antibody (1:800; D7G7 XP Rabbit mAb #8685, Cell Signaling Technology, MA, USA) overnight at 4 °C. Alexa Fluor 488 secondary antibody (1:1000; Anti-Rabbit, Cell Signaling) was added for 1 h. Cells were rinsed and put onto glass slides. Fluorescent images were taken at 4 × by a Zeiss Axiovert 200 M fluorescent microscope.