Anti glial fibrillary acidic protein

Five sections of the brain cortex and anterior horns of the spinal cord from each rat were randomly selected and subjected to immunofluorescence staining. The tissue sections were incubated overnight at 4 °C with anti-NF-200 (1:500; Abcam, Cambridge, MA, USA), anti-GAP-43 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI, USA), anti-BDNF (1:500; Abcam), anti-NF-κB p65 (1:500; Abcam), Anti-p75NTR (1:500; Abcam), anti-MBP (1:500; Abcam), anti-glial fibrillary acidic protein (GFAP, 1:200, Thermo Fisher Scientific, Waltham, MA, USA), anti-COX-2 (1:1000; BioVision, Milpitas, CA, USA), and anti-CD68 (1:100; Santa Cruz Biotechnology) antibodies, individually. After washing in PBS, the sections were incubated with TRITC/FITC-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. The sections were subsequently mounted on glass slides and coverslipped with Antifade Gel Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL, USA). Stained sections were viewed under a microscope at 200x magnification, and images were taken of three fields per tissue section. Areas stained positively for GFAP, MBP, and CD68 were analyzed using NIH Image software. The numbers of cells stained positively for GAP43, caspase-3, NF-200, BDNF, NF-kB p65, COX-2, Caspase-3, and p75NTR were counted.

Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12), Neurobasal Medium, B-27 supplement, phosphate-buffered saline (PBS) buffer, fetal bovine serum (FBS) and anti-glial fibrillary acidic protein (GFAP) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant mouse epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from R&D Systems (Minneapolis, MN, USA). Poly-L-ornithine (PLO), laminin and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Nestin and oligodendrocyte marker O4 (O4) antibodies were purchased from Millipore (Burlington, MA, USA). Anti-Sox2 and Ki67 antibodies were purchased from Abcam (Cambridge, UK). Anti-ionized calcium-binding adaptor molecule 1 (Iba1) was purchased from FUJIFILM Wako Chemicals (Richmond, VA, USA). Growth-factor-reduced Matrigel was purchased from BD Biosciences (Bedford, MA, USA). Alexa 568 goat anti-mouse, Alexa 568 goat anti-rat, Alexa 488 goat anti-rabbit, and Alexa 488 and 568 goat anti-rabbit secondary antibodies were purchased from Life Technologies (Carlsbad, CA, USA). WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH₂) was purchased from Anygen Inc. (Gwangju, Korea).

Primary antibodies include anti-Aβ_1-16 (6E10, Covance Research Products, Emeryville, CA, USA), anti-Aβ_17-24 (4G8, Covance Research Products), anti-p-tau (Thr²³¹, EMD Millipore, Billerica, MA, USA), anti-p-tau (Ser²⁰², AT8, Thermo Fisher Scientific, Waltham, MA, USA), anti-p-tau (Ser⁴⁰⁴), anti-p-tau (Thr¹⁸¹, AT270, AnaSpec, Fremont, CA, USA), anti-total tau (tau46, Cell Signaling Technology, Danvers, MA, USA), anti-synaptophysin (Cell Signaling Technology), anti-GSK3β (Ser⁹, Thermo Fisher Scientific), anti-COX2 (Thermo Fisher Scientific), anti-light chain 3B (LC3B) (Thermo Fisher Scientific), anti-β-tubulin III (Thermo Fisher Scientific), anti-p-synapsin I (Thermo Fisher Scientific), anti- microtubule associated protein 2 (MAP2) (Thermo Fisher Scientific), anti-glial fibrillary acidic protein (GFAP) (Thermo Fisher Scientific), anti- neuronal nuclei (NeuN) (Thermo Fisher Scientific), anti- post synaptic density protein 95 (PSD95) (Thermo Fisher Scientific), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Thermo Fisher Scientific) antibodies. Paired helical filament 1 (PHF1) antibody was kindly provided by Dr. Peter Davies (Albert Einstein University).