Anti gfap

Mice were anesthetized by IP injection of 20% urethane (0.01 ml/g) and perfused transcardially with 0.9% saline and 4% paraformaldehyde (PFA) in PBS (0.01 M, pH 7.4). Immediately after perfusion, brains were dissected and post-fixed in 4% PFA for 24 h, then dehydrated by immersion in a 15% sucrose solution overnight followed by a 30% sucrose solution until the tissue no longer floated in the sucrose solution. Serial coronal/sagittal sections of mPFC and HIPP tissues were cut to a 30 μm thickness using a Leica CM30505 freezing microtome (Leica, Germany). The slices were then treated for membrane permeabilization with 0.5% Triton-X 100 and blocked with 5% BSA in 0.01 M PBS for 1.5 h at RT, followed by incubation with primary antibody against rabbit anti-Iba1 (Wako, 019-19741, 1:1,000) and anti-GFAP (Thermofisher, 13-0300, 1:1,000) overnight at 4°C in 1% BSA/PBS. The following day, slices were given three 10 min washes with PBS and incubated with secondary antibody (Invitrogen) at RT for 2 h followed by three additional washes with PBS for 10 min. Each section was imaged using a 20× lens under a fluorescence microscope (Nikon, Japan). Positive-cell counting was done using Image-Pro Plus (Media Cybernetics, United States) and Image J software (National Institutes of Health, Bethesda, MD, United States).

Immunoblotting analyses of PrP relied on the monoclonal 3F4 antibody (catalog number MAB1562, Millipore Sigma) which recognizes the 109–112 amino acid epitope of human PrP. Antibodies against the NKA subunits consisted of anti-ATP1A1 (catalog number ab7671, Abcam Inc., Toronto, ON, Canada), anti-ATP1A2 (catalog number ab9094-I, Abcam Inc.), anti-ATP1A3 (catalog number MA3-915, Thermo Fisher Scientific) and anti-ATP1B1 (catalog number GTX113390, GeneTex, Irvine, CA, USA). Neuronal and astrocytic markers were detected with the anti-NeuN antibody (catalog number EPR12763, Abcam Inc.), the anti-Tuj-1 antibody (catalog number ab78078, Abcam Inc.) and the anti-GFAP monoclonal (131-17719) antibody (catalog number A-21282, Thermo Fisher Scientific).

The following primary antibodies were used. Mouse monoclonal antibodies anti-histone H2AX phospho-Ser139 (1/1000 Western, 1/200 immunostaining; Millipore-Upstate 05–636, MA, USA), anti-BrdU (1/25 immunostaining; Sigma B8434, UK), anti-WRAP53 (1/200 immunostaining; Abnova H00055135-M04, USA), anti-UBF (1/100 immunostaining; Santa Cruz Biotechnology SC-13125) and anti-B23 (1/100 immunostaining; Abcam ab10530). Rabbit polyclonal antibodies anti-histone H2AX phospho-Ser139 (1/200 immunostaining; Novus Biologicals NB100–384), anti-trimethyl-histone H4 (Lys 20) (1/250 immunostaining; Millipore-Upstate 07–463, USA), anti-53BP1 (1/100 immunostaining; Bethyl Laboratories A300-272A, Inc., USA), anti-CTCF (1/100 immunostaining; Millipore-Upstate 07–729, MA, USA), anti- Iba1 (1/500 immunostaining; Wako 019–19,741), anti-GFAP (1/500 immunostaining; Thermo Fisher Scientific PA3–16727), anti-NeuN (1/100 immunostaining; Abcam ab177487) and anti-Histone H3 (1/2000 Western; Thermo Fisher Scientific PA5–16183). Chicken polyclonal anti-β-galactosidase (1/200 immunostaining; Abcam ab9361). Specific secondary antibody conjugated with FITC or TexasRed were used (Jackson Lab., USA).

Western blot analysis was performed as previously described (Legartova et al., 2013 (link)). In the analysis, we used the following primary antibodies: anti-H3K9 acetyl (#06-942, Upstate-Millipore), anti-histone H3 (#ab1791, Abcam), anti-pan-acetylated-lysine (#ab-21623), anti-HDAC1 (#sc-7872, Santa Cruz Biotechnology, Santa Cruz, CA), anti-HDAC3 (#SAB1404635, Sigma Aldrich), and anti-H4ac (#382160, Merc Millipore, Germany). To evaluate the neuronal differentiation, we used anti-NCAM (#701379, Thermo-scientific), anti-Sox2 (#sc17320, Santa Cruz), anti-GFAP (#13-030, ThermoFisher Scientific), anti-GAPDH (#cs5174S, Cell Signaling), anti-p300 (#05-257 Merc Millipore, Czech Republic), and antibodies against βIII-tubulin (#T5076 Sigma-Aldrich) or Nestin (#ab6142, Abcam). We used the following secondary antibodies for Western blot analysis: anti-rabbit IgG (#A4914, Sigma Aldrich, Czech Republic), anti-mouse IgG1 (#sc-2060, Santa Cruz Biotechnology), and anti-mouse IgG (#A9044, Sigma Aldrich, Czech Republic). The total protein levels were measured using a μQuant spectrophotometer (BioTek Instruments, represented by Biotech, Czech Republic). The data for histone-associated markers were normalized to the total level of histone H3, while the levels of the neuronal markers were normalized to the total GAPDH level.

The brains of rats (n = 4 per group from the second experiment) were removed and post fixed in 4% PFA at 4°C overnight and immersed in 20% sucrose (4% PFA as solvent) followed by 30% sucrose (in 0.1 M PBS). Thirty micrometer thick sections (CM1850, Leica Microsystems, Wetzlar, Germany) were blocked in 2% (wt/vol) BSA (Sigma) and then exposed overnight to the following primary antibody mixtures: anti-GFAP(Thermo, 1:1000) or anti-Iba-1(Wako, 1:1000) at 4°C. After washed five times in PBS, sections were incubated with secondary antibodies (donkey-anti-mouse, Alexa 594 conjugated, 1:1000, Invitrogen, USA; donkey anti rabbit, Alexa 594 conjugated, 1:1000, Invitrogen, USA; Hoechst, 1:1000, Beyotime, China) for 1 h at room temperature in the dark. These sections were rinsed in PBS five times 5 min each and cover slipped in the dark. Sections were imaged at 20× and 40× for analysis, using excitation wavelengths of 633 nm (blue Cy5 labeling), 543 nm (red Cy3 immunofluorescence).

The sections used for immunofluorescence staining were incubated with primary monoclonal mouse anti-MBP antibody (1:500, Abcam, Cambridge, MA), anti-GAP43 and anti-CD68/ED1 (1:100; Santa Cruz, Dallas, TX) antibodies and polyclonal rabbit anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI), anti-NF200 (1:500, Abcam, Cambridge, MA), anti-GFAP (1:200, Thermo Fisher Scientific Waltham, MA), anti-Olig1 (1:500; Abcam, Cambridge, MA), anti-CNTF (1:500, Abcam, Cambridge, MA), anti-NF-kB p65 (1:500, Abcam, Cambridge, MA) antibodies and anti-BDNF(1:500, Abcam, Cambridge, MA) antibodies overnight at 4 °C. The sections were then washed with phosphate-buffered saline (PBS) and incubated with 1:200 TRITC (rhodamine)-conjugated secondary antibodies for 1 h at 37 °C (Invitrogen, Carlsbad, CA). The sections were finally coverslipped with Antifade Gel/Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL).
Five sections from the brain cortex and anterior horns of the spinal cord for each animal were randomly selected and images were photographed under 200x magnification in three vision fields per section. GFAP, MBP, CD68-immunoreactive areas were analyzed with NIH image software, and the numbers of GAP43, caspase-3, NF-200, Olig1, CNTF, NF-kB p65, BDNF-labeled cells were counted.

WT or SOD1-G93A mice of 1, 2, or 4 months old (at least three mice from each experimental group) were sacrificed for immunohistochemistry and perfused with saline followed by 4% paraformaldehyde in phosphate buffer saline (pH 7.4). Sciatic nerves and lumbar spinal cords were collected. Sections (40 μ) of spinal cord tissue and slices (25 μ) of sciatic nerves were prepared as previously described (43 (link)).
Tibial muscles were freshly collected and longitudinally positioned on a plate, covered with OCT, placed in methanol for one minute and after in liquid azote. Neuromuscular junctions (NMJ), from 10 μm slices, were stained with: α-bungarotoxin (αBTX) tetramethylrhodamine (TRITC)-conjugated (1:100, Thermofisher); anti-GFAP (glial fibrillary acidic protein, astrocyte marker) antibody (mouse monoclonal, 1:100, Sigma-Aldrich); anti-calcium channel (a1A-subunit PQ) antibody (rabbit polyclonal, 1:100, Sigma-Aldrich); anti-A20 antibody (mouse monoclonal, 1: 500 Santa Cruz Biotechnology).
For double immunofluorescence staining, we used primary and secondary antibodies as in Table 1. Nuclei were stained with DAPI (1:1,000, Jackson ImmunoResearch).