Mouse anti gfap

Brain sections or cultured astrocytes on coverslips were blocked with 3% bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 for 1 h at room temperature (RT). The sections or cells were then incubated with primary antibodies overnight at 4°C. Two antibodies were added simultaneously for double-immunofluorescence staining. The following antibodies were used: rabbit anti-S100a10 (1:100; Novus, Colorado Springs, CO, United States), mouse anti-C3 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-Iba-1 (1:800; Sigma, United States), mouse anti-GFAP (1:500; Abcam, Cambridge, United Kingdom), and rabbit anti-GFAP (1:500; Abcam). The samples were then incubated with species-specific secondary antibodies conjugated with Alexa Fluor (1:200; Zhuangzhi, Beijing, China) for 2 h at RT, and nuclei were stained with Hoechst-33342 (1:10000, GeneCopoeia, United States). Fluorescent signals were visualized under a confocal laser microscope.

Immunofluorescence procedures were performed as described previously [31 (link), 32 (link)]. Mice were deeply anesthetized and perfused transcardially with 30 mL of 0.9% saline solution (37 °C) followed by 100 mL of 4% paraformaldehyde (Sigma) in 0.1 mol/L phosphate buffer (pH 7.4). Subsequently, brains were removed and kept in 4% paraformaldehyde (Sigma) overnight at 4 °C and then embedded in paraffin. The brains were cut into 5-μm-thick slices, which were incubated with goat anti-Iba-1 (1:500, Abcam), rabbit anti-NeuN (1:200, Millipore), mouse anti-GFAP (1:500, Abcam), or rabbit anti-CHRNA7 (1:200, BOSTER) overnight at 4 °C. Finally, the slices were incubated with the corresponding secondary antibody for 2 h. The sections were analyzed with an Olympus FluoView 1200 confocal microscope system (Olympus Corporation, Tokyo, Japan), and photomicrographs of representative fields were taken.

Differentiated cells in alginate beads were fixed in
4% paraformaldehyde and 70% ethanol for 30 minutes.
Samples were then permeabilized with 2%
Triton X-100 (Sigma, St.Louis, MO, USA) for 30
minutes. Blocking in 1 mg/ml BSA and incubating
primary antibodies against mouse anti-NESTIN
(1:300, Abcam, Cambridge, MA, USA), mouse anti-
GFAP (1:600, Abcam, Cambridge, MA, USA) and
mouse anti-MAP2 (1:300, Abcam, Cambridge, MA,
USA) were performed overnight. The secondary antibody,
anti-mouse FITC-conjugated IgG antibody
(1:500, Abcam, Cambridge, MA, USA), was used
for 2 hours at 37˚C. For nucleus visualization, cells
were stained with diamidino-2-phenylindole (DAPI,
1:1000, Sigma, St.Louis, MO, USA). For negative
control, primary antibody was eliminated. To merge
the pictures, image J software1.42 software (Wayne
Rasband, National Institutes of Health, Bethesda,
MD, USA) was used. Hundred cells were counted per
sample.

Immunofluorescence staining and western blotting were performed according to a previous study [20 (link)]. For immunofluorescence staining, the following primary antibodies were used: rabbit anti-TIMP1 (1:50, Proteintech), mouse anti-NeuN (1:50, Millipore) and mouse anti-GFAP (1:50, Abcam), and the following secondary antibodies were used: fluorescein isothiocyanate (FITC)-conjugated donkey anti-rat IgG (1:100, Proteintech) and Alexa Fluor 555-conjugated donkey anti-mouse IgG (1:100, Beyotime). For western blotting, the following primary antibodies were used: rabbit anti-TIMP1 (1:1000, Proteintech) and rabbit anti-GAPDH (1:1000, Proteintech), while horseradish peroxidase (HRP)-goat anti-rabbit immunoglobulin G (1:3000, Proteintech) was used as the secondary antibody.

The rats were anesthetized on PDN 30 with 100 mg/kg sodium pentobarbital (i.p.) and cardiac perfusion was performed with 0.9% saline. Some of the brains in each group were extracted for immunofluorescence staining as described in our previous study (Zhao et al., 2018 (link)). The primary antibodies and dilution criterions were as follows: ab654, rabbit anti-GLUT4, 1:200; ab177487, mouse anti-NeuN, 1:400; ab7260, and mouse anti-GFAP, 1:800 (Abcam, United States). The secondary antibodies were as follows: A-21145, Alexa Fluor 594, A-11008, and Alexa Fluor 488 (1:1000, Molecular Probes, Invitrogen). All sections were incubated with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, United States) for 15 min while avoiding light.

As previously described [40 (link), 41 (link)], rats (n = 8 per group) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and perfused with PBS followed by fresh 2% glutaraldehyde and 4% paraformaldehyde through the ascending aorta. The brain was collected and fixed in 4% paraformaldehyde for 4 h and then dehydrated in 30% sucrose overnight at 4°C. The brain tissues were embedded in the optimal cutting temperature compound and finally cut to a thickness of 15 μm in a -20°C cryostat for immunofluorescent staining. After blocking in 10% normal goat serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the tissue sections were incubated overnight at 4°C in 10% normal goat serum in PBS containing primary antibodies such as rabbit anti-pCREB, 1 : 150, Cell Signaling Technology; rabbit anti-BDNF, 1 : 100, Abcam; mouse anti-Neun, 1 : 300, Abcam; mouse anti-GFAP, 1 : 500, Abcam; and mouse anti-Iba1, 1 : 500, Abcam. Afterwards, these slices were incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies and Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies (1 : 600, Jackson ImmunoResearch) for 1 h. Then, they were washed in PBS and cover-slipped with VECTASHIELD Mounting Medium with DAPI (Cat: H-1200, Vector Lab). Images were captured by a microscopic imaging system (Olympus BX61 and FluoView).