Anti gfap

Cells grown on coverslips were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 for 10 min, and blocked with 1% bovine serum albumin (BSA) and 10% Normal Donkey Serum(NDS) or 10% Normal Goat Serum(NGS) for 1 h at RT^{4 (link)}. The following primary antibodies were then used to incubate with the cells overnight at 4 °C, the neuronal markers: anti-NFM, anti-MAP2, anti-TuJ-1 (1:200, Abcam), anti-NSE and anti-GFAP(1:200, Neuromics, MN, USA); cell surface markers: anti-CD29, CD44 (1:100, Abcam) and CD71, CD73 (1:100, Santa Cruz, CA, USA); the epigenetic markers: anti-histone acH3K9(1:200, Santa Cruz), anti-histone meH3K9(1:200, Abcam), anti-phosphor-Histone H3S10(1:1,000, Santa Cruz); the pluripotent markers: anti-Oct4, anti-Sox2 and anti-Nanog (1:200, Cell Signaling Technology, Danvers, MA, USA)^{4 (link), 18 (link)}. After that, the cells were incubated with CY3/488/543-labeled secondary antibody (Invitrogen, CA, USA) for 1 h, and the results of immunofluorescence were observed under confocal microscopy. The catalog numbers for all primary antibodies used in this research are presented in Table S1.

The cell culture plastic ware was from Greiner BioOne. The DMEM/F12 culture media, B27 Supplement (without antioxidants) and L-Glutamine were from Life Technologies. Poly-L-Lysine (Mol. Weight ≥ 300.000) and Triiodo Thyronine were from Sigma. The human PDGF-AA, human IGF-1 and human EGF were all recombinant and from Peprotech (cat. # 100–13A, 100–11 and AF-100–15, respectively). The recombinant human bFGF was a gift from Dr. Baldi (IBYME, Argentina) and the recombinant human PDGF-BB was a generous gift from Laboratorios Beta S.A. (Argentina). Fetal Calf Serum was purchased from Natocor (Argentina) and Heparin was from Nortia S.A. (Argentina). Antibodies; anti NG2 (Chemicon), anti PDGFRα (Neuromics), anti MBP (gift from former Campagnoni Lab, UCLA, USA), anti GFAP (Neuromics) and anti βTubulin III (Sigma). Further details on antibodies are listed in Table 1. The peptides sequences of PDGF proteins were the following:
PDGF-AA variant 1:
SIEEAVPAVC KTRTVIYEIP RSQVDPTSAN FLIWPPCVEV KRCTGCCNTS SVKCQPSRVH HRSVKVAKVE YVRKKPKLKE VQVRLEEHLE CACATTSLNP DYREEDTGRP RESGKKRKRK RLKPT.
PDGF-BB:
SLGSLTIAEP AMIAECKTRT EVFEISRRLI DRTNANFLVW PPCVEVQRCS GCCNNRNVQC RPTQVQLRPV QVRKIEIVRK KPIFKKATVT LEDHLACKCE TVAAARPVT.

Whole brains were separated from the skull, fixed overnight with PFA, and then placed in a 30% sucrose/0.05 M PBS solution at 4 °C. The brains were cryo-sectioned using a Cryostat (Microsystems AG, Leica, Wetzlar, Germany) with 30 μm thick coronal sections and stored in a cryoprotectant solution consisting of 0.2 M PB, 25% ethylene glycol, 25% glycerol, and water at 4 °C [29 (link)]. All sections were collected in six different series and processed for immunostaining as previously described [30 (link)].
Brain coronal cryosections (30 μm in thickness) containing the hypothalamus were incubated with rabbit anti-glial fibrillary acidic protein antibody (anti-GFAP, 1:5000; Neuromics, Edina, MN, USA) for astrocytes or rabbit anti-ionized calcium-binding adaptor molecule 1 antibody (anti-Iba-1, 1:1000; Wako, Osaka, Japan) for microglia [31 (link)]. Then, it was stained with biotinylated goat anti-rabbit IgG secondary antibody and detected with the avidin-biotin complex (ABC) standard kit (Vector Laboratories, Burlingame, CA, USA). Then, 0.5 mg/mL 3,3′-diaminobenzidine (Sigma, St. Louis, MO, USA) in 0.003% H₂O₂/0.1 M PBS was added to sections to visualize the antibody-positive signals. To quantify GFAP- or Iba-1-positive cells, the images of stained brain sections were taken by a microscope (Olympus Optical, Tokyo, Japan).