Glial fibrillary acidic protein (gfap)

For western blotting, cell lysates were generated by resuspending cells in RIPA buffer (Sigma), then sonicated as previously described (Hearst et al., 2010 (link)). Lysates were subjected to sodium dodecyl sulfate– polyacrylamide gel electrophoresis (4–20% acrylamide precast gels; Bio-Rad Laboratories, Hercules, CA, United States) as described earlier (Hearst et al., 2010 (link)). Equal amounts of proteins were loaded in each well. Proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad), blocked for 1 h with blocking solution (1X TBS, 0.1% Tween-20 with 5% w/v non-fat dry milk) and incubated overnight with primary antibodies: Casp-3 and βIII Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, United States), GFAP and ACTB (Cell Signaling Technology, Danvers, MA, United States). Western blots were imaged using Alexa-794 secondary antibodies (Santa Cruz) and the Odyssey Infrared Imaging System (LI-COR Biosciences, NE, United States). Western blots were analyzed using Image J software, as previously described (Hearst et al., 2010 (link)). The average ratios from protein band optical densities were obtained from three independent experimental repeats.

Mice were euthanized by intra-cardiac perfusion and brains were fixed in neutral buffered formalin. Paraffin-embedded brain sections were assessed with the following antibodies: GFAP (Cell Signaling, 1:1000), Iba1 (Wako, 1:1000); pSer129/81A αSyn (1:3000; [13 (link), 36 (link)]); EP1536Y (AbCam, 1:1000); p62 (Protein Tech; 1:1000), cd11b (AbCam; 1:250), MHCII (Novus; 1:50) and NeuN (Abcam; 1:500). For all antibodies except cd11b and MHCII, antigen retrieval was performed by steaming for 25 min in water. For cd11b and MHCII, antigen retrieval was done by steaming in Dako Target Retrieval Solution S1699 (modified citrate buffer, pH 6.1). Colorimetric slides were treated with ImmPress reagents (Vector Labs) and visualized with 3, 3’diaminobenzidine followed by hematoxylin counterstaining. Fluorimetric slides were visualized with Alexa Fluor conjugated secondary antibodies (Invitrogen) and counterstaining with 4’, 6-diamidino-2-phenylindole (DAPI; Southern Biotech). All colorimetric slides were scanned using the Aperio XT whole slide scanner while fluorescent slides were visualized using Olympus BX60 microscope with a color digital camera.

Whole-cell lysates of tumor cells were processed for SDS-PAGE and blotting according to standard methods, as described previously [24 (link)]. Western blots were probed with antibodies against AC133 (Miltenyi Biotec), SOX-2 (R&D Systems), GFAP (Cell Signaling), actin (Santa Cruz Biotechnology), and HRP-conjugated secondary antibodies (Dianova).

Both tissue and cell samples were lysed using RIPA buffer supplemented with protease inhibitor cocktail (Sigma, USA). Protein concentration was measured using Bradford reagent (Sigma, USA). 20–30 μg of protein samples were loaded and separated using SDS-PAGE followed by wet transfer on a methanol charged PVDF membrane. The membrane was blocked using 5% Bovine serum albumin for an hour at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies used at 1:1000 concentration were mouse monoclonal hTDP-43 (Abnova, Taiwan), LC3 (Novus Biologicals, USA), Beclin-1 (Novus Biologicals, USA), p62 (Millipore, USA), ATG-5 (Millipore, USA), and Actin (Millipore, USA), GFAP (Cell signalling technologies, USA),. Subsequently, the blots were incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibodies. Moreover, the blots were developed using ECL detection reagents and visualized with a StarBright Blue 520 (Bio-Rad Laboratories, USA). All band intensities were quantified using the ImageJ lab software. The membranes were incubated with suitable peroxidase conjugated secondary antibodies (Vector Laboratories, USA). Once the incubation was over, PBST wash was given to the blots. Blots were then treated with ECL reagent and developed using UNITECH imaging system (Cambridge) from Millipore, CA USA.

Eyes with the attached optic nerve segment, surgically removed from perfused mice, were post-fixed in 4% PFA. Retinas were dissected out for either whole-mount preparations or cryosections. The optic nerve was separated from the eye and cut longitudinally with a Leica cryostat. Retinal whole-mounts or sections were blocked in the staining buffer containing 5% normal donkey serum and 0.1% Triton X-100 in PBS for 1 hr before incubation with primary antibodies. Primary antibodies used: Tuj1 (Covance, 1:500, Princeton, NJ), PTEN (Cell Signaling Technology, 1:250, Danvers, MA), p-AKT (Cell Signaling Technology, 1:200), AKT (Cell Signaling Technology, 1:250), p-GSK3α (Abcam, 1:250, UK), p-GSK3β (Cell Signaling Technology, 1:400), GSK3b (Cell Signaling Technology, 1:250), p-S6 (Cell Signaling Technology, 1:200), p-eIF2Bε (EMD Millipore, 1:300, Billerica, MA), GAP43 (obtained from Dr. Larry Benowitz, 1:500), CSPG (Sigma, 1:200, St. Louis, MO), and GFAP (Cell Signaling Technology, 1:200). Secondary antibodies used: DyLight Cy3/594/647-conjugated AffiniPure antibodies (Jackson ImmunoResearch, 1:500, West Grove, PA). Confocal images were acquired using a Zeiss LSM 510 EXCITER microscope. Fluorescence channel colors were switched for co-localization studies if necessary. Images were analyzed and organized using ImageJ and Photoshop.