Immunofluorescence was carried out as described previously [32] (link), [33] (link). The cellular localization of TLR3 was measured in brain sections from a cohort of mice (n = 3 per group) killed at 24 h after preconditioning; sham-operated mice served as controls. The mice were perfused transcardially with saline followed by 4% paraformaldehyde. The brain region –1.0 mm from the optic chiasm was then cut into 30-µm coronal sections by a cryostat (SM2000R, LEICA, Berlin, Germany). For double staining of TLR3 and glial fibrillary acidic protein (GFAP), the sections were incubated together with goat anti-TLR3 antibody (1∶50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-GFAP antibody (1∶200; Epitomics, Cambridge, UK) overnight at 4–8°C, and then incubated sequentially with fluorescein-labeled secondary antibody for 2 h at room temperature. Finally, images in the brain cortex were observed with a fluorescence microscope (Olympus BX-51; Olympus Optical, Tokyo, Japan).
Mouse anti gfap antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Mouse anti-GFAP antibody is a primary antibody that specifically recognizes the Glial Fibrillary Acidic Protein (GFAP), a major intermediate filament protein found in astrocytes and other glial cells. This antibody can be used to detect and visualize GFAP in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5 2 confocal laser scanning microscope, by Leica (2 mentions)
Anti rabbit antibody conjugated with alexa fluor 488, by Cell Signaling Technology (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Sm2000r, by Leica (1 mentions)
Goat anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti tlr4 antibody, by Abcam (1 mentions)
Mouse anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti lc3b antibody, by Abcam (1 mentions)
Rabbit anti caspase 3 antibody, by Abcam (1 mentions)
Mouse anti neun antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Fitc conjugated goat anti mouse igg, by Vector Laboratories (1 mentions)
Tritc conjugated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Rabbit anti gr antibody, by Cell Signaling Technology (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Anti α synuclein, by Abcam (1 mentions)
Mouse anti gfap cy3 conjugated antibody, by Merck Group (1 mentions)
8 protocols using mouse anti gfap antibody
1
Immunofluorescence Analysis of TLR3 Expression
2
Protein Quantification and Western Blotting
3
Autophagy Induction in Neurotoxicity Model
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SVGA cells were seeded at 0.8 × 106 on 1.5 mm cover slips followed by treatment with METH and gp120 IIIb. After termination of the treatment, the cells were fixed with 1 : 1 ice-cold methanol and acetone solution for 20 min at –20 °C. The wells were air dried followed by blocked and permeabilized with 1% BSA in PBS with 0.1% Triton for 30 min. After blocking, the cells were then incubated with a cocktail of rabbit anti-LC3B antibody (1 : 2000) and a mouse anti-GFAP antibody (1 : 1500) (Abcam, Cambridge, MA, USA) overnight in a humidified chamber. After three washes with 0.1% Triton in PBS, the cells were incubated in the dark chamber for 1 h with an anti-mouse antibody conjugated with Alexa Fluor 555 (1 : 2000) and an anti-rabbit antibody conjugated with Alexa Fluor 488 (1 : 2000) (Cell Signaling Technology) followed by three washes with 0.1% Triton in PBS. Finally, the cover slips were transferred onto glass slides with 10 μl of Vectashield mounting reagent with DAPI. The microscopy analysis was performed using a Leica TCS SP5 II laser scanning confocal microscope. The images were captured using a 40X zoom lens and, ImageJ software was used to analyze the images and calculate the intensity. GFAP was used as housekeeping protein to normalize the intensity of the LC3II.
4
Investigating Enteric Glial Cell Apoptosis
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The HGNC database (https://www.genenames.org/ ) was applied to transform the obtained JCD target genes into mouse genes. The Joint-Pathway analysis of MetaboAnalyst 5.0 was used to enrich differentially expressed metabolites (DEMs) and target genes associated with JCD. Except for those nominated by other diseases, the top 25 pathways were selected for integrated analysis. The degree of apoptosis of enteric glial cells (EGCs) in colon tissue was analyzed by immunofluorescence. Rabbit anti-caspase3 antibody (Abcam; 1:100) was applied overnight at 4 °C, and mouse anti-GFAP antibody (Abcam; 1:100) was stained at 37 °C for 30 min followed by 4’,6-diamidino-2-phenylindole for 10 min at room temperature. ImageJ graphics processing software was used to calculate the apoptosis of EGCs. Five fields of each slide were randomly observed, and densitometric values were estimated.
5
Quantification of Autophagy in Cocaine-Treated SVGA Cells
6
Immunofluorescent Staining of Serine Racemase
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Frozen sections (20-μm thick) were prepared for the detection of SR expression by immunofluorescent staining. Tissue sections were incubated with the following primary antibodies at 4°C overnight: rabbit anti-SR antibody (1:100, Santa), mouse anti-GFAP antibody (1:1000, Abcam), rabbit anti-SR antibody (1:500, Abcam), rabbit anti-D-serine antibody (1:1000, Abcam), and mouse anti-NeuN antibody (1:1000, Chemicon). After rinsing with PBS, the sections were incubated with corresponding secondary antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 488 (Jackson Immunoresearch) for 2–4 h at room temperature and protected from light. For the staining of SR, a competitive peptide was used as a control to ensure the specificity of the anti-SR antibody. The nuclei were counterstained by DAPI (Sigma, St Louis, MO, United States).
7
Immunostaining of Astrocyte GR and GFAP
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For immunofluorescent assay, the cultured astrocytes were fixed with 4% paraformaldehyde in 10 mM phosphate-buffered saline (PBS; pH 7.4) for 20 min at room temperature, blocked with 3% normal goat serum (Sigma-Aldrich) for 30 min at room temperature, and incubated with rabbit anti-GR antibody (1:100, monoclonal, Cell Signaling, Boston, MA, USA) and mouse anti-GFAP antibody (1:200, monoclonal, Abcam, Cambridge, UK) overnight at 4°C. Then, the cells were washed with PBS, incubated with TRITC-conjugated goat anti-rabbit IgG (1:200; Vector, Burlingame, CA, USA) and FITC-conjugated goat anti-mouse IgG (1:200; Vector) secondary antibodies for 60 min while shaking at room temperature in darkness, and washed with PBS. Finally, the astrocytes were incubated for 2 min with nucleic acid stain, Hoechst33258 (Invitrogen, Carlsbad, CA, USA), diluted 1:5000 in PBS. All primary and secondary antibodies were diluted in PBS containing 1% normal goat serum. The images were captured using a confocal laser scanning microscopy (LSM 710, Carl Zeiss, Germany).
8
Immunocytochemistry Techniques for Visualization of Cellular Targets
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For single immunocytochemistry of the free floating sections, the antigen-antibody complex was visualized by conventional Nickel intensified DAB reaction product as described previously (Vereczki et al., 2006) . For fluorescence single, double and triple immunocytochemistry the sections were processed by ABC (ABC Elite Kits, Vector Laboratories, Burlingame, CA) and biotin amplification as described previously (Berghorn et al., 1994) . The antibodies used were as follows: rabbit anti-mGluR1a (a generous gift of Dr. Shigemoto), dilution for Ni-DAB at 1:30,000, and for biotin amplification fluorescence staining at 1:15,000; mouse anti-GFAP antibody at 1:50,000 (Abcam, Cambridge, UK); mouse anti-NeuN (Chemicon Interna
tional, Temecula, CA) at 1:7000 for fluorescence staining; mouse anti-GFAP-Cy3 conjugated antibody (Sigma-Aldrich) at 1:50,000 and anti-α synuclein at 1:50,000 (Abcam).
tional, Temecula, CA) at 1:7000 for fluorescence staining; mouse anti-GFAP-Cy3 conjugated antibody (Sigma-Aldrich) at 1:50,000 and anti-α synuclein at 1:50,000 (Abcam).
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