Rabbit anti gfap antibody

Routine immunocytochemistry was performed as follows: Cultures were fixed in 4% paraformaldehyde for 12 min. After washing three times (3 min each) with PBS, the non-specific binding of the antibodies was blocked using tween-20 PBS (PBST) blocking buffer (with Glycine and Bovine Serum Albumin). The cells were then incubated overnight at 4 °C with a primary antibody solution prepared in PBS with 3% goat serum and 0.5% Triton X-100 for permeabilization. The primary antibodies used were mouse anti-β-tubulin-III for neurons and mouse anti-GAD65 for GABAergic neurons (both from DSHB), as well as rabbit anti-GFAP antibody (Sigma) for astrocytes, rabbit anti-MAPII antibody for mature neurons, rabbit anti-VGLUT1/2 for glutamatergic neurons, and rabbit anti- NGN2 for motor neurons (all from Cell Signaling). The next day, after washing out the unbound primary antibodies with PBS three times, appropriate secondary antibodies were added—AlexaFluor488 goat-anti-mouse and AlexaFluor594 goat-anti-rabbit (Invitrogen). Finally, the cells were stained with DAPI. The appropriately stained cells were visualized by fluorescence microscopy using BioTekCytation-5 Bioimager/Plate Reader (Winooski, VT, USA). Gen−5 analysis software (3.08 BioTek, Winooski, VT, USA) was used to count and quantify the different cell populations, unbiased, based on fluorescence.

For immunofluorescence, cells plated on chamber slides were fixed with 4% Paraformaldehyde at room temperature for 15 min. Following washing three times with PBS, cells were blocked by PBS containing 0.3% Triton X-100 and 10% goat serum for 1 h. Then cells were incubated in primary antibody solution overnight at 4°C. The primary antibody used was rabbit anti-GFAP antibody (1:1,000, Sigma). After three times washing with PBS, cells were incubated with goat anti-rabbit secondary antibody (1:2,000) for 1 h at room temperature. After washing the secondary antibody, 0.5 ng/ml of DAPI (Sigma) was added in the chamber for 30 min to stain the cell nuclei. The cells were examined with a digital eclipse 80i microscope (Nikon, Tokyo, Japan). 5-ethynyl-2′-deoxyuridine (EdU) staining was achieved by the Click-iT Plus EdU Alexa Fluor 488 Imaging kit (Thermo Fisher Scientific, Waltham, MA, USA) and performed according to manufacturer’s instructions.

Immunostaining was performed as previously described [29 (link)]. Briefly, E16.5 mouse cortical neurons were dissociated and seeded on an 8-well chamber and cultured in vitro for 7 days (DIV7). The neurons were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min and permeabilized with 0.2% TritonX-100 in PBS for 10 min. After being blocked with 5% Normal Goat Serum (Thermo Fisher) at room temperature (RT) for 1 h, the cells were incubated with mouse anti-Tuj1 antibody (Cat# 801201, Biolegend, San Diego, CA, USA) and rabbit anti-GFAP antibody (Sigma, New York, NY, USA, HPA056030) at 4 °C overnight. Then the cells were incubated with Cy3 conjugated anti-rabbit IgG (A10520, Invitrogen) and Alexa Fluor 488 conjugated anti-mouse IgG (A10680, Invitrogen) secondary antibodies at RT in darkness for 1 h. After washing 3 × 5 min with 1 × PBS, cells were then mounted with DAPI-Fluoromount-G™ Clear Mounting Media (Cat# 010020, Southern Biotech, Birmingham, AL, USA). Fluorescent images were acquired using a confocal microscope.

To show the morphological evidence of the changes in neurons, astrocytes, and microglia in the hippocampus, immunohistochemical staining was conducted for NeuN, GFAP, and Iba-1, respectively, as previously described [20 (link),57 (link)]. In addition, proliferating cells and neuroblasts were visualized with the immunohistochemistry of Ki67 and DCX. Briefly, animals (n = 5 per group) were anesthetized with a mixture of 75 mg/kg alfaxalone and 10 mg/kg xylazine on the 56th day of diet feeding, and blood was obtained by cardiac puncture in the right ventricle. Thereafter, animals were perfused transcardially, and the brain was coronally sectioned with a 30 μm thickness between 2.0 and 2.7 mm caudal to the bregma based on gerbil stereotaxic coordinates [58 (link)]. Four sections located 150 μm apart were selected and incubated with each antibody; mouse anti-NeuN antibody (1:1000; Merck Millipore, Temecula, CA, USA), rabbit anti-GFAP antibody (1:1000; Merck Millipore), rabbit anti-Iba-1 (1:500; Wako, Osaka, Japan), rabbit anti-Ki67 (1:1000; Abcam, Cambridge, UK), or rabbit anti-DCX (1:2000; Abcam). Immunoreaction was visualized with 3,3′-diaminobenzidine tetrachloride (Sigma, St. Louis, MO, USA) in 0.1 M Tris-HCl buffer (pH 7.2). Sections were dehydrated and mounted on gelatin-coated slides in Canada balsam (Kanto Chemical, Tokyo, Japan).

Immunohistochemical staining for NeuN, GFAP, and Iba-1 was performed to visualize surviving neurons, astrocytes, and microglia in the hippocampus 4 d after ischemia. The animals were re-anesthetized with isoflurane and perfused transcardially with physiological saline and 4% paraformaldehyde via the left ventricle. The part of the brain located on the gerbil atlas 1.4–2.0 mm caudal to the bregma [42 (link)] was sectioned at a thickness of 30-μm using a sliding microtome (HM430, Thermo Scientific, Waltham, MA, USA). Five sections, at 90-μm intervals from each other, were incubated with mouse anti-NeuN antibody (1:1000; EMD Millipore, Temecula, CA, USA), rabbit anti-GFAP antibody (1:1000; EMD Millipore), and rabbit anti-Iba-1 antibody (1:500; Wako, Osaka, Japan) for 48 h at 4°C. After sequential treatment with biotinylated goat anti-mouse IgG or anti-rabbit IgG and streptavidin–peroxidase complex (1:200; Vector, Burlingame, CA, USA) for 2 h at 25°C, immunoreactions were developed using 3,3-diaminobenzidine tetrachloride (Sigma).