Immunofluorescence analysis in cultured cells

Cells were fixed in 4% PFA (paraformaldehyde) for 10 min and then permeabilized by incubating cells with 0.2% TX-100 for 5 min at 25 °C, unless stated otherwise. After blocking with 10% goat serum for 1 h, primary antibodies were added in the same 10% goat serum buffer and incubated overnight at 4 °C. Antibodies recognized phosphorylated STAT3 (Tyr705, 1:50, Cell Signaling Technology 9145), BDNF (1:40, Santa Cruz Biotechnology sc-65514), TrkB (1:40, Santa Cruz Biotechnology sc-377218), DPP4 (1:100, Cell Signaling Technology 67138), FN1 (1:50, Abcam ab2413), and THBS1 (1:50, Abcam ab85762).

Fluorescent signals were detected by adding fluorescent secondary antibodies [Invitrogen, Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568; and Goat anti-Rabbit IgG (H + L), Superclonal Recombinant Secondary Antibody, Alexa Fluor 488] in 10% goat serum buffer (1:1000) for 1 h at 25 °C. Finally, DAPI was used to counterstain the nuclei; images were taken using a fluorescence microscope (BZ-X Analyzer, Keyence) and analyzed with ImageJ. The intensity of BDNF immunofluorescence was calculated using ImageJ by analyzing the fluorescent signal present within an area of 10 µm radius from the center of the DAPI-stained nuclei. A total of 60 cells were analyzed per experimental group (20 from each replicate).