Ab 11333

Cells cultured from the palatal mucosa and gingival graft were characterized as fibroblasts by their morphology and staining with fibroblast surface protein (FSP; ab 11333, Abcam, Cambridge, UK) by means of immunostaining [11 (link)]. Cells were seeded in an 8-well plate with 1×10⁴ cells per well and incubated at 37°C with 5% CO₂ during 18 hours to adhere in subconfluence. Subsequently, the wells were divided into groups (control, Pg LPS, Ec LPS and negative control, which was performed without the primary antibody) in duplicate, to cell stimulation. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour. Coverslips were mounted with mounting medium VECTASHIELD Hard-Set Mounting Medium containing 49,6-diamidino-2-Phenylindole (DAPI; H-1500, Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscope laser scanning (TCS model, SPE, Leica Mannheim, Germany).