Mab5564

Eyeballs were dissected and fixed in 4% paraformaldehyde (Cat. No. BM-698; Bostonbioproducts, BioProducts, Ashland, MA) for 2 hours at room temperature followed by immersing into 20% sucrose solution in phosphate buffered saline (PBS) for 2 hours. The eyeballs were embedded in O.C.T. compound (Cat. No. 4583; Sakura Finetek USA, Inc., Torrance, CA) on dry ice. Frozen sections of the retina (10 μm) were incubated with a blocking buffer containing 4% bovine serum albumin and 0.5% Triton X-100 in PBS for 1 hour followed by incubation with a primary antibody against β-III-tubulin (1:500; Cat. No. MAB5564, Millipore), Recoverin (1:500; Cat. No. AB5585, Millipore) or H3K27me3 (1:500; Cat. No. 9733S, Cell Signaling Technology, Danvers, MA) in the blocking buffer for overnight at 4°C. Slides were washed with PBS 3x at 10 min. each before incubation with Cy3/Cy2-conjugated secondary antibody in the blocking buffer [Cy3-AffiniPure Donkey Anti-Mouse (1:500; Cat. No. 715-165-151, Jackson ImmunoResearch) or Cy2-AffiniPure Donkey Anti-Rabbit (1:500; Cat. No. 711-095-152, Jackson ImmunoResearch)] were applied for 1 hour at room temperature. Slides were washed with PBS 3 x at 10 min. each. The slides were mounted in a mounting media containing DAPI (Cat. No. H-1200; Vector Laboratories Inc.) and imaged with a TSC SP5 confocal microscope (Leica Microsystems, Richmond, IL).

For SDS-PAGE and Western blot analysis, a total of 25 DRG explants per well were seeded in 12-well plastic plates (Grenier). For protein harvesting, cultures were washed with PBS, and DRGs were scraped into 90 μl of sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 m Tris-HCl, pH ∼6.8). Samples were boiled for 5 min, centrifuged, and stored at −80°C for later analysis. Antibodies used for immunoblotting were: anti-βIII-tubulin (Millipore MAB5564, 1:10,000), anti-neurofilament M (Millipore AB1987, 1:1000), anti-caspase-3 (NEB 9662, 1:1000), anti-TrkA (Millipore 06-574, 1:1000), anti-TrkB (Millipore 07-225, 1:1000), anti-TrkC (Millipore 07-226, 1:1000), and the previously described anti-p75NTR (Barker and Shooter, 1994 (link)).

RGCs were isolated from postnatal day three Sprague-Dawley pups as described^{61 (link)} and seeded immediately in neurobasal-SATO (nb-SATO) media at 3,000 RGCs/well in 96-well plates (Falcon 087723B, Corning Inc, Corning, NY) coated with poly-D-lysine (70 kDa, 10 μg/ml, Sigma-Aldrich Corp., St. Louis, MO) and laminin (2 μg/ml, L-6274, Sigma-Aldrich Corp., St. Louis, MO). RGCs were cultured at 37 °C, 10% CO₂ for 3 days with MBV (0-80 µg/ml) or UBM-ECM (250 μg/ml). Using a Live/Dead Imaging Kit (R37601, Life Technologies, Carlsbad, CA), the number of viable RGCs per area was quantified^{14 (link)}. Five random, non-overlapping fields were imaged at 20×, totaling 45 fields of view. For total neurite growth, RGCs were labeled with anti-βIII tubulin (1:300, TUJ-1, MAB5564, Millipore, Burlington, MA). The first ten RGCs encountered, not contacting another RGC, were measured, totaling 90 RGCs per group, as described^{62 (link)}.

Antibodies directed against β-III tubulin (Tuj1, 1:10,000 for immunofluorescence, MAB5564) were purchased from Millipore (Canada). The TRPV1 inhibitor capsazepine (CPZ) was obtained from Tocris Biosciences and was used at a final concentration of 10 μM. The TRPV1 agonist capsaicin (Sigma Aldrich, Canada) was used at 1 μM. The L-type channel inhibitor nifedipine was purchased from Sigma Aldrich used at 10 μM. NAD⁺ (Sigma Aldrich, Canada) and N-acetyl-L-cysteine (NAC, Sigma Aldrich, Canada) were used at final concentrations of 5 mM and 20 mM, respectively. EGTA (VWR, Canada) was used at 6 mM. The mitochondrial ROS scavenger MitoQ (a generous gift of Dr. Michael Murphy) was used at 1 μM (Kelso et al., 2001 (link)). CCCP (Sigma Aldrich, Canada) was used at a final concentration of 50 μM.

DRG explants were fixed in 4% paraformaldehyde solution in PBS for 15 min, washed once in PBS and blocked in 5% milk in Tris-borate buffer and 0.3% Triton X-100 for 1 h at room temperature (RT). Explants were incubated overnight at 4°C with mouse monoclonal antibody against βIII-tubulin (Millipore, MAB5564) diluted 1:10,000 in blocking solution. DRGs were washed twice in PBS and then incubated with goat anti-mouse conjugated to Alexa Fluor 488 (Jackson ImmunoResearch, 115-545-003) diluted 1:5000 in blocking solution for a minimum of 3 h at RT. Explants were imaged using a Zeiss ObserverZ.1 inverted epifluorescence microscope with an automated motorized stage (5× magnification with tilling). From a stitched master image of the plate generated by Zen 2 software (Zeiss), quarter DRG fields were cropped to generate a set of images for analysis using the R script program Axoquant 2.0 (Johnstone et al., 2018 (link)). Final measurements were plotted as the mean axonal area of DRGs from three embryos. Increments of 500 μm were used for statistical analysis (normalized to same increments in control condition).