Immunofluorescent Analysis of FGF-21 and BMP7

Skin specimens were fixed in 4% paraformaldehyde and were then dehydrated and embedded in paraffin. Paraffin sections were successfully dewaxed in different gradients of the dewaxing solution, absolute ethanol, and distilled water. The dewaxed sections were placed in EDTA antigen repair buffer (pH 8.0) for antigen repair and blocked with a 10% donkey serum block. Primary antibodies were then added: anti-rabbit-FGF-21 (1:200) and BMP7 antibody (1:200), and incubated overnight at 4°C. On the next day, the slides were washed three times with PBS (pH 7.4) in a rocker device, the objective tissue was covered with a secondary antibody (responding appropriately to the primary antibody in the species), and was incubated at room temperature for 50 min in dark conditions. Finally, DAPI dye solution was added and incubated for 10 min. The images were observed and collected under a fluorescence microscope (GE Bioscience, Newark, NJ, USA).