Ab6673

For immunostaining, tissues were fixed in Zn-formalin for 24 hr and embedded in paraffin. Sections were deparaffinized, rehydrated, and prepared by antigen retrieval for 6 min each, and then blocked in 5% donkey serum for 1 hr at room temperature, incubated with either Rabbit anti-BNIP3 antibody at 1:100 (A5683, Abclonal), Rabbit anti-NF-kB-p65 1:400 (8242S, Cell Signaling Technology), Rabbit anti-Phospho-c-jun 1:200 (3270S, Cell Signaling Technology), Rat anti-ki67 1:100 (14-5698-82, ebiosciences), Rabbit anti-pRB-Ser807/811 1:400 (8516, Cell Signaling Technology), anti-p21 1:1000 (ZRB1141, Sigma), Rat anti-Phospho H3 1:1000 (H9908, Sigma), or Goat anti-GFP (ab6673, Abcam) at 1:250 overnight at 4°C, washed, incubated with secondary antibody AF594-anti-rabbit (A-21207, Invitrogen), Alexa594 anti-rat (A-21209, Invitrogen), and Alexa488 anti-goat (11055, Invitrogen) antibodies at 1:200 for 1 hr at room temperature, counterstained with DAPI, washed, and mounted. Slides were visualized and imaged using an Olympus IX71 inverted multicolor fluorescent microscope and a DP71 camera using ×400 magnification. MFI was calculated by measuring average intensity over a fixed threshold for all images. NF-kB staining was imaged via Zeiss LSM880 confocal microscope using ×63 oil-immersion objective, and intensity was measured on 0.8 µm z-sections.

After behavioral experiments, we performed transcardial perfusion with PBS followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were post-fixed in 4% paraformaldehyde for an additional 12–18 h at 4° C. Before sectioning, brains were rinsed three times in PBS and embedded in 4% agarose-PBS. Then, 50-μm-thick slices were made using a vibrating microtome (Leica, VT100S). Sections were then suspended in blocking solution (10% Normal Goat Serum and 0.1% Triton-X100 in 1× PBS) for 1 h followed by overnight incubation at 4 °C with the primary antibody. Next, sections were washed with PBS, incubated for 1 h at room temperature with the secondary antibody at 1:500 dilution. For histological visualization of GCaMP6s, we used primary goat polyclonal anti-GFP antibody (1:500 dilution; Abcam, ab6673) and secondary donkey anti-goat Alexa Fluor 488 (1:500 dilution; Abcam, ab150129). Sections were then dry-mounted on slides using Vectashield (Vector Labs, H1000) before imaging. No immunostaining was performed for the visualization of FusionRed or tdTomato. Imaging was performed using an upright fluorescence macroscope (Olympus BX61). Images were acquired using Ocular Scientific Image Acquisition Software (Teledyne Imaging). Visualization and analysis were performed using ImageJ/FIJI software packages.

For immunohistofluorescence staining, rats were anesthetized and transcardially perfused with 200 mL 0.9% saline, followed by 400 mL 4% paraformaldehyde at seven days post-transplantation (Supplementary Fig. 3a). Thereafter, the spinal cord was dissected (1.0 cm above and below the injured site, Supplementary Fig. 3b) and cryoprotected in 30% sucrose in 0.1 M PBS dehydrate at 4 °C. The tissues were sliced at 10 mm thickness by cryostat and fixed on the PLL-coated slides. The details of immunohistofluorescence staining were consistent with the above-mentioned description (Fan et al. 2019 (link)). The primary antibodies included anti-p75 (ab245134, 1:200, Abcam), GFP (ab6673,1:200, Abcam), Iba1 (019-19741, 1:500, Wako), iNOS (ab49999, 1:200, Abcam), Arg-1 (ab60176, 1:200, Abcam), NF68 (2835s, 1:50, Cell Signaling Technology) and DAPI (ab228549, 1:1000, Abcam). Other following procedures were same as the immunocytofluorescence.

Primary fly neurons and fibroblasts were fixed in 4% paraformaldehyde (PFA) in 0.05 M phosphate buffer (PB; pH 7–7.2) for 30 min at room temperature (RT); for anti-Eb1 staining, ice-cold +TIP fix (90% methanol, 3% formaldehyde, 5 mM sodium carbonate, pH 9; stored at −80°C and added to the cells; Rogers et al., 2002 (link)) was added for 10 mins. Adult brains were dissected out of their head cases in PBS and fixed with 4% PFA in PBS for 1 hr, followed by a 1 hr wash in PBT.
Antibody staining and washes were performed with PBT. Staining reagents: anti-tubulin (RRID:AB_477593, clone DM1A, mouse, 1:1000, Sigma; alternatively, RRID:AB_2210391, clone YL1/2, rat, 1:500, Millipore Bioscience Research Reagents); anti-DmEb1 (gift from H. Ohkura; rabbit, 1:2000; Elliott et al., 2005 (link)); anti-Elav (mouse, 1:1000, DSHB, RRID:AB_528218); anti-GFP (ab6673, goat, 1:500, Abcam, RRID:AB_305643); Cy3-conjugated anti-HRP (goat, 1:100, Jackson ImmunoResearch); F-actin was stained with Phalloidin conjugated with TRITC/Alexa647, FITC or Atto647N (1:100 or 1:500; Invitrogen and Sigma). Specimens were embedded in ProLong Gold Antifade Mountant.