For immunofluorescence staining, tissue sections were washed with 0.1 M PBS for 10 min at room temperature. After rinsing with PBS, tissue sections were blocked with 10% normal goat serum in 0.1 M PBS containing 0.3% Triton X-100 for 2 h at room temperature and then incubated with the following primary antibodies overnight at 4°C, rabbit anti-SphK1 (Abcam, Cambridge, MA, United States; used at 1:500), mouse anti-SphK1 (Santa Cruz, United States; used at 1:500), mouse anti-S1PR2 (Santa Cruz Biotechnology, CA, United States; used at 1:500), rabbit anti-GFAP (Abcam, Cambridge, MA, United States; used at 1:1000), and rabbit anti-NeuN (Abcam, Cambridge, MA, United States; used at 1:1000). On the following day, tissue sections were washed three times for 10 min in 0.1 M PBS and then incubated for 2 h at room temperature with donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488, Abcam, United States; used at 1:500), and goat anti-mouse IgG H&L (Alexa Fluor® 647, Abcam, United States; used at 1:500) conjugated secondary antibodies. Following this incubation period, tissue sections were washed three times with 0.1 M PBS for 10 min and then mounted with Vectashield DAPI Hardset mounting medium (Solarbio, China). Immunofluorescence staining pictures were captured employing a LSM800 confocal microscope (Zeiss, Germany).
Rabbit anti gfap
Manufactured by Abcam
Sourced in United States, United Kingdom, Japan
Rabbit anti-GFAP is a primary antibody that recognizes the Glial Fibrillary Acidic Protein (GFAP), a cytoskeletal protein found in astrocytes and other glial cells. This antibody can be used for the detection and analysis of GFAP in various applications, such as immunohistochemistry, western blotting, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti neun, by Abcam (12 mentions)
Rabbit anti iba1, by Abcam (10 mentions)
Rabbit anti iba1, by Fujifilm (9 mentions)
Mouse anti neun, by Merck Group (8 mentions)
Goat anti iba1, by Abcam (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Mouse anti β actin, by Merck Group (4 mentions)
Mouse anti nestin, by Abcam (4 mentions)
Rabbit anti shh, by Santa Cruz Biotechnology (4 mentions)
Mouse anti gfap, by Merck Group (4 mentions)
Rabbit anti sox2, by Abcam (3 mentions)
Mouse anti neun, by Abcam (3 mentions)
Rabbit anti neun, by Merck Group (3 mentions)
Rabbit anti map2, by Abcam (3 mentions)
Rabbit anti il 1β, by Abcam (3 mentions)
Rat anti brdu, by Abcam (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Alexa fluor 647, by Abcam (2 mentions)
Goat anti iba1, by Fujifilm (2 mentions)
74 protocols using rabbit anti gfap
1
Immunofluorescence Staining of SphK1, S1PR2, GFAP, and NeuN
2
Immunocytochemical Analysis of Astrocytes
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The subculture of astrocytes was planted in Petri dish containing DMEM/F12 complete medium and cultured for 24 h. Astrocytes were fixed in 4% paraformaldehyde for 30 min, washed with 0.01 mol/L phosphate-buffered saline (PBS) three times, permeated by 0.02 % TritonX-100 for 3 min, washed with 0.01 mol/L PBS three times. Astrocytes were incubated with rabbit anti-GFAP (1:1000, Abcam, United States) at 4°C overnight, followed by incubations with FITC-conjugated goat anti-rabbit IgG (1: 1000 in PBS, Sigma, United States) for 90 min. At last, DAPI (1:2000, Abcam) was applied for 5 min to label the nuclei of all astrocytes. After staining, Petri dishes were examined using a Leica SP2 confocal microscope (Leica, Germany).
3
Astrocyte and Microglia Immunostaining Protocol
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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.
4
Spinal Cord Protein Expression Analysis
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Animals were sacrificed after deeply anesthetized with isoflurane, total proteins from the lumbar enlargements of spinal cords were extracted by ice-cold Radio Immunoprecipitation Assay (RIPA) lysis. Proteins were separated on SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, United States). After blocking with 5% nonfat milk, the samples were incubated with rabbit anti-β-actin (1:3000, Abcam, United States), rabbit anti-GAPDH (1:3000, Merck Millipore, United States), mouse anti-VGluT2 (1:2000, Abcam, United States), goat anti-Iba-1 (1:2000, Wako, Japan), rabbit anti-GFAP (1:2000, Abcam, United States), rabbit anti-BDNF(1:2000, Abcam, United States), or rabbit anti-TrkB (1:2000, Abcam, United States) at 4 °C overnight. The next day, the corresponding rabbit, goat, or mouse Horseradish peroxidase (HRP)-conjugated secondary IgG (1:20000, Jackson ImmunoResearch, United States) was incubated for 1 h at room temperature. The gray value of the protein on the membrane of the corresponding molecular size was detected using western blotting detection kit (WesternBright Sirius, Advansta, United States) and ChemiDoc XRS imaging system (Bio-Rad). Image Lab 3.0 system (Bio-Rad) was utilized to perform standardized analysis on the test results.
5
Western Blot Analysis of Neural Markers
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Western blot analysis was performed as described previously [35 (link)], with slight modifications. Briefly, cells were lysed on ice with radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and sonicated to reduce sample viscosity. Protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA), equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Western blotting was performed using the following antibodies at 4 °C overnight: rabbit anti-β-III-tubulin (1:5000; Covance); rabbit anti-MAP-2 (1:2000; Millipore); rabbit anti-GFAP (1:5000; Abcam); rabbit anti-CNPase, rabbit anti-JNK, rabbit anti-p-JNK, rabbit anti-c-Jun, rabbit anti-p-c-Junser63, and rabbit anti-p-c-Junser73 (all 1:1000; all from Cell Signaling Technology); and mouse anti-β-actin (1:10,000; Sigma-Aldrich). Blots were incubated with horseradish peroxidase-labelled secondary anti-rabbit and anti-mouse antibodies, and immunoreactive bands were visualized on film by enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA) (all 1:10,000, Abcam). Western blots were quantified with ImageJ software from three independent experiments. The intensities of the bands were normalized to β-actin.
6
Immunohistochemical Analysis of Spinal Cord Injury
7
Immunofluorescence Characterization of Cell Cultures
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For immunofluorescence studies, the cells were grown on sterile coverslips in 24-well plates. On specified days, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 minutes, permeabilized with 0.1% Triton X-100 for 30 min and then blocked with 5% FBS in PBS for 30 min at room temperature. The fixed cells were incubated with mouse anti-MAP2 (1∶500; Abcam, Cambridge, UK), mouse anti-NeuN (1∶1000; Abcam, Cambridge, UK), rabbit anti-Sox2 (1∶250; Abcam, Cambridge, UK), rabbit anti-CD11b (1∶500; Abcam, Cambridge, UK), rabbit anti-GFAP (1∶500; Abcam, Cambridge, UK) and mouse IgM anti-O4 (1∶100; Sigma, USA) primary antibodies for 1 hr. The cells were then incubated with FITC-, Texas Red- and Cy5-coupled secondary antibodies (1∶200; Abcam, Cambridge, UK) for 1 hr. Hoechst 33342 was used as a nuclear stain. Images were viewed and analyzed using LSM710 confocal imaging software (Carl Zeiss MicroImaging Inc, Germany).
8
Immunohistochemical Analysis of Brain Markers
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Brain sections were permeabilized with 0.3% Triton X-100 for 10 min and then washed. Nonspecific staining was blocked with 3% BSA for 1 h. The sections were incubated with primary antibodies overnight at 4 °C. Primary antibodies included rabbit anti-PGC-1α (1:300, Abcam), goat anti-Iba-1 (1:800, Abcam), mouse anti-eGFP (1:250, Abcam), rabbit anti-Iba-1 (1:500, Wako), rabbit anti-GFAP (1:1000, Abcam), rabbit anti-NeuN (1:500, Abcam), rabbit anti-NLRP3 (1:50, Abcam), rabbit anti-ASC (1:50, Santa Cruz), goat anti-GFAP (1:1000, Abcam), and mouse anti-NeuN (1:1000, Abcam). After incubation with primary antibodies, the sections were washed with PBS (5 × 5 min) and then incubated with appropriate secondary antibodies at room temperature for 1 h. For the analysis of neuronal apoptosis, brain sections were incubated with TUNEL reagents following the manufacturer’s guidelines.
9
Immunohistochemical Profiling of Aged Mouse Brain
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Cryostat, 25 μm free-floating sections were fixed in 4% paraformaldehyde/50 mM for 15 min and then washed extensively with PBS. For phenotyping, the tissue was incubated with: (i) macrophages marker, rabbit anti-ED1 (1:1,000, Abcam, UK); or (ii) the neuronal nuclei marker, rabbit anti-NeuN (1:1,000, Millipore, Germany); or (iii) the astrocytic marker, rabbit anti-GFAP (1:1,000, Abcam, UK) at 4°C overnight. The next day, sections were rinsed with PBS and incubated with Alexa Fluor® 488 goat anti-rabbit IgG or Alexa Fluor® 647 goat anti-rabbit IgG for the aged mice brains. After final rinsing, sections were brought to Superfrost Plus slides and mounted using PVA/DABCO-containing medium.
10
Magnetic Field-Induced Neuronal Differentiation
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ADMSCs (1 × 104 cells) were seeded onto the substrates in 48‐well plates and exposed to a rotating MF for 5, 10, and 15 days. After fixation with 4% paraformaldehyde for 10 min, the cells were permeabilized with 0.1% Triton X‐100 in PBS for 10 min, followed by washing in PBS three times. Samples were then blocked with 5% BSA for 1 h. To evaluate neuronal differentiation, antibodies against neuronal markers were used. These samples were incubated with primary antibodies at 4 °C overnight. Primary antibodies included mouse anti‐nestin (marker of NSCs), mouse anti‐Tuj1, rabbitanti‐MAP2 (neuronal specific markers), and rabbit anti‐GFAP (marker of astrocyte), respectively (Abcam). Next, the samples were washed three times with PBS and then incubated with appropriate secondary antibodies (Alexa Fluor 488 conjugated goat anti‐rabbit or Alexa Fluor 546 conjugated goat anti‐mouse IgG) for 1 h at room temperature. After washing three times in PBS, the cells were stained with DAPI (Invitrogen) for at least 10 min. Fluorescence images were acquired using a laser confocal microscope.
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