Gfap antibody

Paraformaldehyde was purchased from Merck and D-Saccharose was from Roth, Karlsruhe, Germany. Alexa Fluor conjugated secondary antibodies and ProLong^® Gold antifade reagent from Invitrogen/Life Technologies Europe, Darmstadt, Germany. Zonulin 1 antibody was purchased from Thermo Fisher Scientific (Invitrogen) (Waltham, MA, USA); Claudin3 antibody from Abcam (Berlin, Germany); FABP2 antibody from Thermo Fisher Scientific (Invitrogen); LPS antibody from Origene (Rockville, MD, USA); and IL6 antibody was purchased from Cell signaling Technologies (Danvers, MA, USA); GFAP antibody was purchased from Sigma Aldrich (St. Louis, MO, USA); Cytokeratin and Vimentin antibody from Abcam. For SHANK3 western blotting in-house polyclonal rabbit SHANK3 antibodies were used that have been described previously [27 (link),52 (link)]. iScriptTM cDNA Synthesis Kit, SSoAdvanced Universal SYBR^® Green Supermix and customized PrimePCR plates were purchased from Bio-Rad, Hercules, CA, USA. QuantiTect Primer Assays, RNeasy Mini Kit and QuantiFastTM SYBR_Green RT-PCR kit were purchased from Qiagen, Hilden, Germany. Unless otherwise indicated, all other chemicals were obtained from Sigma-Aldrich.

Immunofluorescence staining and double/ triple immunostaining were performed as previously described [12 (link)]. Briefly, the polyacrylamide hydrogel (PAG) tissues were harvested and cryosectioned at 35 μm. The sections were first incubated with 5% normal donkey serum in 0.1 M PBS for 2 h at room temperature. The sections were then incubated overnight at 4 °C with the following primary antibodies: CXCL1 antibody (1:100, rabbit; Boster), phosphor-NF-кB p65 (Ser536) (pNF-кB) antibody (Rabbit, 1:500; Sigma), CXCR2 antibody (1:100, rabbit; Abcam), GFAP antibody (1:1000, mouse; Sigma), CD11 antibody (1:250, mouse; Abcam), and NeuN antibody (1:300, mouse; Abcam). The sections were then incubated for 1 h with FITC- or Cy3-conjugated secondary antibodies (1500, Abcam) at room temperature. For double immunofluorescence, sections were incubated with a mixture of primary antibodies at 4 °C overnight, followed by a mixture of FITC- and Cy3-conjugated secondary antibodies. Sections were coverslipped using a water-based mounting medium containing DAPI (Bioss, China). The stained sections were examined using an Olympus fluorescence microscope, analyzed by Image Pro-plus 6.0 (Image Pro-Plus Kodak, USA), and images were taken with a CCD Spot camera. Negative controls in which the primary antibody was omitted or replaced with PBS were used to confirm immunospecificity.

After passage, the cells were seeded on cover slips which processed by 100 μg/mL poly-L-lysine and incubated with differentiating medium which contains 100 × N2 supplement, 100 × B27 supplement and 1% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) in DMEM/F12 (without b-FGF). At the end of 7 day cultures, cells were harvested for differentiation analysis. A total of 5–7 randomly selected images were taken in each well, and the number of cells expressing β-tubulin III, GFAP tested by β-tubulin III antibody (Sigma-Aldrich Inc. St. Louis, MO, USA) and GFAP antibody (Sigma-Aldrich Inc. St. Louis, MO, USA) were counted (at least 200 cells each test case), and data were collected from at least three independent experiments.

Western blotting was used to measure retinal GFAP levels in WT and Pparα^-/- mice at 8, 15, and 40 weeks of age as described previously [25 (link)]. Mice were euthanized with carbon dioxide asphyxiation and retinas were snap-frozen in liquid nitrogen. One retina per animal was homogenized in Holt’s lysis buffer, and 10 μg of protein were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were probed with GFAP antibody (Sigma; Cat # G3893; RRID AB_477010) and subsequently reprobed with HRP-conjugated β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat # sc-47778 HRP; RRID AB_2714189). Band density was quantified using Alpha Imager software (San Jose, CA, USA). N is expressed as number of retinas per genotype, per time point.

Cortical cultures were prepared from brains of embryonic day 17–18 (E17/E18) embryos from timed pregnant Wistar rats (Charles River), as previously reported40 (link). In brief, cortex was dissected out in Hanks’ balanced salt solution buffered with Hepes and dissociated via trypsin treatment. Cells were plated at 1 × 10⁶ cells on 3.5-cm dishes precoated with poly-L-lysine. After 2 days of culturing in neurobasal medium with B-27 supplement (0.5 mM L-glutamine, 1% antibiotic penicillin/streptomycin), half of the medium was changed every 3–4 days. All experimental treatments were performed on 12-day “in vitro” (DIV) cultures in Neurobasal + ½ B27 fresh medium. The culture cell composition was examined using immunocytochemical staining for neurons (NeuN antibody, Sigma, 1: 200), astrocytes (GFAP antibody, Sigma 1:400) and microglia (Iba1 antibody, Abcam 1: 200) with DAPI nuclear staining. Mixed cultures contain about 50% NeuN⁺ cells, 45% GFAP⁺ cells and 4% of Iba1⁺ cells.

At 3 days after TBI, the injured mice were perfused with chilled PBS and then with 4 % paraformaldehyde. The brains were immersed in paraformaldehyde for 24 h and subsequently dehydrated with ethanol and permeated/embedded with paraffin. Six-micrometer-thick sections were cut with a cryotome and mounted on charged slides. Four sections from the injury epicenter for each brain were later deparaffinized, subjected to antigen retrieval by boiling in citrate buffer for 20 min, and blocked with 5 % goat serum for 1 h at room temperature. They were then incubated overnight at 4 °C in anti-ionized calcium-binding adaptor molecule 1 (Iba-1) (catalog number 019-19741; Wako Chemicals, Richmond, Virginia, USA) or anti-glial fibrillary acidic protein (GFAP) antibody (catalog number G9269; Sigma-Aldrich, Saint Louis, MO, USA) solution followed by secondary antibody tagged with Alexa Fluor 488 (Thermo Fisher, Waltham, MA, USA). The fluorescence was visualized with a ×20 objective and quantified using the MetaMorph software (Molecular Devices Inc., Sunnyvale, CA, USA). Cells were counted in the peri-contusional cortex in three non-overlapping fields per section and the corresponding fields for the contralateral cortex.

Cells were seeded on glass coverslips. At the end of incubation (1, 4 or 24 h) with 10 µM of fluorescent labeled mApoE-PA-LIPs, neural cultures were fixed with paraformaldehyde (PFA), washed three times with protein-free PBS and incubated with anti-βIII Tubulin antibody (1:250; Promega Corporation, Fitchburg, WI, USA) or with glial fibrillary acidic protein (GFAP) antibody (1:400; Sigma-Aldrich Co) for 2 h at room temperature and, after washes, with Alexa Fluor 594-conjugated goat anti-mouse IgG (1:100; Thermo Fisher Scientific) for 1 h at room temperature. 1 µM DAPI (Sigma-Aldrich Co) was used to label nuclei. Images were acquired with LSM710 inverted confocal laser scanning microscope equipped with a Plan-Neofluar 63×1.4 oil objective (Carl Zeis Meditec AG, Jena, Germany). Excitation wavelengths were λ=488 nm to detect LIPs, λ=610 nm to detect βIII Tubulin or GFAP, and λ=405 nm to detect nuclei.