Immunofluorescent Analysis of Smad2/3 in Gingival Fibroblasts

Immunofluorescent analysis was performed as recently described^{23 (link)}. Briefly, human gingival fibroblasts were plated onto Millicell® EZ slides (Merck KGaA, Darmstadt, Germany) and treated with PRF 30% for 30 min. Cells were then fixed in paraformaldehyde and blocked in 5% BSA and 0.3% Triton in PBS at room temperature for 1 hour. Cells were subsequently incubated with Smad2/3 antibody (1:800; D7G7 XP® Rabbit mAb #8685, Cell Signaling, MA, USA) overnight at 4 °C. Alexa Fluor 488 secondary antibody (1:1000; Anti-Rabbit, Cell signaling Technology, USA) was applied for 1 hour. Cells were washed and mounted onto glass slides. Fluorescent images were captured at 10x and 100x in oil immersion using a Zeiss Axiovert 200 M fluorescent microscope.