Alexa fluor 568 or 647

Animals were perfused with 4% paraformaldehyde in 0.1 M PBS, pH 7.4, at three, five and 28 days. Spinal cord segments containing the lesion site, or whole brain from pHrodo-myelin injected mice, were removed and processed for cryostat sectioning (20-μm-thick cross sections). Immunofluorescence was performed using rat anti-CD11b (1:250; Serotec, Raleigh, NC), rabbit anti Iba1 (1:500; Wako, Richmond, VA), anti-rat CD86 (1:200; BD Bioscience, Mississauga, ON), rabbit anti-P2ry12 (1:500; kindly provided by Dr. Oleg Butovsky), rabbit anti-Tmem119 (1:1 hybridoma supernatant; kindly provided by Dr. Mariko Bennett, Ben Barres Lab), rabbit anti-Ki67 (1:500; Abcam, Cambridge, MA), and chicken anti-GFP (1:500; Abcam, Cambridge, MA) and detected using the appropriate secondary antibodies at 1:500; anti-rabbit Alexa Fluor 568 or 647, anti-rat Alexa Fluor 568 or 647, and anti-chicken Alexa Fluor 488 (Invitrogen, Carlsbad, CA). All images were visualized using a confocal laser scanning microscope (FluoView FV1000; Olympus) using FV10-ASW 3.0 software (Olympus) and prepared with ImageJ. Histochemical staining with Luxol fast blue was used to assess myelin loss 28 days after injury. Myelin was quantified as a measure of Luxol fast blue (mean gray value, ImageJ) across the whole cross section, measured at 200-μm intervals over the 2-mm length of the cord.

Tissue collected were fixed for 2 h at room temperature in 4% PFA. This was followed with 3x wash of 1x PBS (Amresco, Solon, Ohio, USA). Tissues were then infused under a sucrose gradient for cryo-protection before cryo-embedding in Tissue-Tek^® O.C.T. Compound (Sakura Finetek, Torrance, California, USA). Prior to antigen staining, cryo-sections were permeabilized in 0.5% TritonX-100 (Chem Supply, Gillman, South Australia) before blocking with 20% normal goat serum. In this study, primary antibodies used includes rat anti-mouse CD31 (1:100), rabbit anti-αSMA (1:200), rabbit anti-FSP1 (1:200), rabbit anti-SLUG (1:200), rabbit anti-SOX9 (1:500), rabbit anti-ERG (1:500) and mouse anti-SOX9 (1:200). Primary antigen staining was conducted overnight at 4 °C. Sections were then subjected to 3 × 5 min washes in 1x PBS + 0.1% Tween-20 (Amresco, Solon, Ohio, USA). Secondary antibodies conjugated with Alexa-fluor 568 or 647 (Invitrogen, Carlsbad, CA, USA) were used for fluorescence detection. Sections were incubated with secondary antibodies for 40 min at room temperature. Nuclear staining was revealed in specimens mounted with ProLong^® Gold mounting media containing DAPI (Invitrogen, Carlsbad, CA, USA).

S2 cells were plated on concanavalin A-coated MatTek (Ashland, MA) dishes (for live cell imaging) or #1.5 coverslips (for fixed-cell imaging) and allowed to adhere for 30 minutes. Samples to be fixed were washed in 1X phosphate-buffered saline (PBS) then fixed with -20°C 100% MeOH for 15 minutes. Samples were stained at room temperature with primary antibodies in PBS+5% Normal Goat Serum (NGS) for 1 hour and with secondary antibodies in PBS+5% NGS for 30 minutes. Primary antibodies used were guinea pig anti-Asterless (1:30,000; G. Rogers, University of Arizona Cancer Center, University of Arizona, Tucson, AZ), anti-α-tubulin (1:500, clone DM1A; Sigma-Aldrich). Secondary antibodies were Alexa Fluor 568 or 647 (1:1000; Life Technologies). Coverslips were mounted using Aqua Poly/Mount (Polysciences, Inc., Warrington, PA) [24 (link)].