Alexa fluor 568 conjugated goat anti rabbit igg

At 28 days following the operation, the sciatic nerve tissues containing the area of crush injury site (5 mm away from the sciatic notch) were harvested and fixed with 4% paraformaldehyde. Then the longitudinal sections and transverse sections of the nerve tissue were prepared. Moreover, the sections were stained with NF200 (CST, 1:200), S100β (Abcam, 1:200), TuJ1 (Abcam, 1:200), MBP (Abcam, 1:200), CD31 (Abcam, 1:200), CD34 (Abcam, 1:200), VEGFR (Abcam, 1:200), GAPDH (Abcam, 1:200), Akt (Abcam, 1:500), p-AKT (Abcam, 1:500), PI3K (Abcam, 1:500), p-PI3K (ThermoFisher, 1:500) and PTEN (Abcam, 1:500). Secondary antibodies were as follows:Alexa Fluor568–conjugated Goat Anti-Rabbit IgG (Abcam), CoraLite594-conjugated Goat Anti-Mouse IgG (Proteintech, China), CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech), CY3-labeled goat anti-rabbit (Servicebio) and AlexaFluor594-labeled goat anti-rabbit IgG (Abcam). Besides, we also used FITC-Tyramide (Servicebio) and CY3-Tyramide (Servicebio) to amplify fluorescence intensity. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy. The percentages of the markers positive areas were calculated by dividing integrated option density by selected region area, then multiplied by 100%. All parameters were measured using ImageJ.

Slices of mouse striatal tissue were blocked with 5% goat serum (ImmunoReagents, USA), and incubated with primary antibodies against TH (1:500, Santa Cruz) overnight at 4 ◦C. Then, Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:1000, Abcam) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) were incubated at room temperature for 1 h. Next, sections were stained with 4′,6-diamidino-2′ -phenylindole (Sigma, USA). Finally, the fluorescence changes were observed through a fluorescence microscope, green fluorescence intensity represents the expression level of TH- positive cells (Olympus, Japan).