Shandon m1 embedding matrix

At the completion of experimental testing, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and transcardially perfused with 4% paraformaldehyde in 0.1 M PBS (pH 7.5). The spinal cord was removed and post-fixed overnight, cryoprotected in 30% sucrose for 48 hours, blocked, and frozen at −80°C in Shandon M1 embedding matrix (Thermo Fisher Scientific; Waltham, MA). Spinal tissue was sliced at 50 μm using a cryostat, slide mounted and stained for Nissl and myelin similar to previous studies^{37 (link),43 (link)}. Photomicrographs were taken at 150 μm intervals from approximately C5 to T2.

Freshly dissected eyes were fixed in 4% MEMPFA (0.1 MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO₄, 4% paraformaldehyde) overnight at 4°C, infiltrated with 30% sucrose in PBS for 24 hours. For each of the time points, both right (crush) and the left (control) eyes from each of two or three WT individuals were cryo-embedded into the same mold using Shandon M-1 embedding matrix (Thermo Scientific Inc.), stored at −80°C and cryosectioned at 14 μm thickness. Hybridization was carried out with hydrolyzed digoxigenin labeled RNA probes transcribed from cDNAs as described previously (Zhang et al., 2008 (link)) (see Table S1), most of which were commercially available (Open Biosystems and ThermoFisher). Detection was carried out using a fluorescent peroxidase substrate, cy3-tyramides (Perkin Elmer). Quantification of the uchl1 in situ hybridization signal in retina sections was carried out by manually segmenting the retinal ganglion cell layer and half of the inner plexiform layer based on the DAPI signal in an average of two sections per retina. This was followed by multiple segmentation based quantifications as previously described (Mills et al., 2015 (link)), reporting values for the most representative fold-change at the given statistical significance value, as determined by a 2-tailed Student’s t-test.

Mice were perfused with 20 ml cold PBS containing 10U heparin/ml followed by 30 ml of 4% PFA in PBS. The brains were removed from the skull, incubated successively with 4% PFA in PBS (16h, 4°C) and 20% sucrose in PBS (16h, 4°C), embedded in Shandon M1 embedding matrix (Thermo Scientific, Waltham, MA) and cut into 30 μm sections. Floating sections were stained using the antibodies to phosphorylated neurofilament heavy protein epitope (NFHP) (mouse IgG1; Covance, New York, NY), myelin basic protein (MBP) (mouse IgG2b; AbCam, Cambridge, MA), ionized calcium binding adaptor molecule 1 (Iba1) (rabbit IgG; Wako Chemicals, Richmond, VA), platelet-derived growth factor receptor-α□□PDGFRα □ (goat IgG; R&D Systems, Minneapolis, MN), NeuN (mouse IgG1; Millipore, Billerica, MA), glial fibrillary acidic protein (GFAP) (mouse IgG2b; BD, Franklin Lakes, New Jersey) and CSF-1R (IK) (rabbit IgG; produced in our laboratory). Secondary antibodies, conjugated to either Alexa 488, Alexa 594 or Alexa 647, were from Life Technologies (Grand Island, NY). Images were captured using an Olympus Bx51 upright fluorescence microscope with Olympus MicroSuite Five Biological software. Quantification of cell numbers was performed manually. Areas were measured using Image J software (imagej.net). Images were cropped and adjusted for brightness, contrast and color balance using Adobe Photoshop CS4.