Cyanine3 (cy3)

Spine cord tissues were fixed in 4% paraformaldehyde and dehydrated using 30% sucrose overnight. After embedding into OCT compound (Tissue Tek), tissues were cut into 16 μm section. Sections were blocked using 5% normal goat serum and then were incubated with the diluted primary antibody specific to F4/80 (ab111101, Abcam, MA, USA), CD16/32 (Catalog # 14-0161-82, Invitrogen, Waltham, MA, USA), and Arg1 (sc-271430, Santa Cruz, USA), overnight at 4 °C. Cy3 or FITC-conjugated secondary antibody (Santa Cruz) were incubated with sections at room temperature for 1 h. For cellular immunofluorescence, cells were fixed with 4% paraformaldehyde and incubated with the primary antibody specific to F4/80 and CD11b (ab8878) and then incubated with Cy3 or FITC-conjugated secondary antibodies. DAPI (C1002, Beyotime, China) was used to stain the nucleus in tissue sections and cells before capturing images. The images were acquired using a fluorescence microscope (Nikon, Japan).

The RPCs were plated onto glass cover slips. Primary antibodies were used to characterize the cells (PAX6, 1:100, Santa Cruz; CRX, 1:50, Santa Cruz; Nestin, 1:500, BD Bioscience; Sox2, 1:1000, Chemicon; GFAP, 1:1000, Chemicon). Cy3 (1:1000, Santa Cruz) was used as the secondary antibody and the slides subsequently imaged using a confocal microscope (Leica TCS NT, Leica Microsystems).

Monoclonal or polyclonal antibodies (anti-mouse type I collagen and anti-rat CD68, Santa Cruz, California, USA) and secondary antibodies labeled with FITC (Sigma Co., St. Louis, Missouri, USA) or Cy3 (Santa Cruz, California, USA), and DAPI stain (Daiko, Japan). All other reagents were of analytical grade (Roche, Germany).