Alexa fluor 488 goat anti rabbit igg h l

To assess the angiogenic effect of the M2 macrophage exosomes on the SC of the rats post-SCI, the proportion of proliferating blood vessels was detected by immunofluorescence staining. After SCI for 3 days, the SC tissues were collected and fixed with paraformaldehyde (4%) for 3 h. After incubation with 6% sucrose in PBS overnight, an optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) was used to embed the tissues and then sectioned at 5 μm thickness (n = 5/group). After air-drying for 15 min, 10% normal goat serum/PBS was added to the sections for 1 h, and then, they were exposed to proliferating cell nuclear antigen (PCNA, Shanghai, China) rabbit monoclonal antibody (1:200; Abcam, Cambridge, UK) or CD31 mouse monoclonal antibody (1:100; Abcam, Cambridge, UK), Nestin (1:200, Abcam, Cambridge, UK), NeuN (1:200, Abcam, Cambridge, UK), and Sox2 (1:200, Sangon Biotech, Shanghai, China) overnight at 4 °C. After washing with PBS, the sections were exposed to the secondary antibody Cy3 goat anti-mouse IgG (H + L) (1:1000, Jackson, Bar Harbor, ME, USA) or Alexa Fluor 488 goat anti-rabbit IgG (H + L) (1:1000, Jackson, Bar Harbor, ME, USA) for 30 min. The sections were rinsed thrice with PBS and exposed to DAPI solution (CST, Boston, MA, USA) for 15 min. The number of positive cells in the SC was counted.

Paraffin sections were dewaxed, rinsed with gradient alcohol, and placed in 0.1 mol/L citrate repair solution (pH 6.0) for antigen repair. After washing, the tissues were circled with an immunohistochemical pen and incubated with a drop of 5% goat serum at room temperature for 1 h. Two primary antibodies at appropriate concentrations were added dropwise, and a mixture of secondary antibodies [Coralite 594 goat anti-mouse IgG (H+L) (Proteintech, Cat. No. SA00013-3) and Alexa Fluor 488 goat anti-rabbit IgG (H+L)] (Jackson ImmunoResearch, Cat. No. 115-545-003) were added dropwise. The sections were incubated for 30 min at room temperature and washed. Nuclei were stained with DAPI (Erwan Pathology, Cat. No. ER201707132) that was added dropwise.