All substances were purchased from Sigma‐Aldrich (Taufkirchen, Germany) unless stated otherwise. Papain was obtained from Roche (Mannheim, Germany). Chloromethyl‐tetramethyl‐rosamine (Mitotracker Orange), NP‐EGTA (o‐nitrophenyl EGTA), NPE‐ATP (P(3)‐[1‐(2‐nitrophenyl)]ethyl ester of ATP), and Fluo‐4 AM were from Molecular Probes (Life Technologies, Carlsbad, CA). For immunocyto‐ and histochemical staining, the following primary antibodies were used: rabbit anti‐Kir4.1 (1:200; Sigma‐Aldrich), mouse anti‐glial fibrillary acidic protein (GFAP; 1:200; G‐A‐5 clone, Sigma‐Aldrich), goat anti‐calretinin (1:500, Swant, Marly, Switzerland), mouse anti‐calbindin (1:400, Swant), rabbit anti‐PKCα (1:300, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti‐cellular retinaldehyde‐binding protein (CRALBP; 1:300, Santa Cruz), rabbit‐anti‐Iba1 (1:500, Wako Chemicals, Neuss, Germany), and mouse anti‐glutamine synthetase (1:1000, Merck Millipore, Darmstadt, Germany). As secondary antibodies, we used Cy5‐conjugated donkey anti‐goat, Cy3‐conjugated donkey anti‐rabbit, Cy2‐conjugated donkey anti‐mouse, Cy3‐conjugated goat anti‐rabbit, and Cy2‐conjugated goat anti‐mouse. All secondary antibodies were obtained from Dianova (Hamburg, Germany) and applied at 1:200 dilution. Apoptosis was detected by the in situ cell death detection kit (Roche, Mannheim, Germany).
Mouse anti glial fibrillary acidic protein
Manufactured by Merck Group
Sourced in United States
Mouse anti-glial fibrillary acidic protein (GFAP) is a lab equipment product that serves as a marker for astrocytes, a type of glial cell in the central nervous system. It is used to identify and study these cells in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Lsm510 meta nlo, by Zeiss (3 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Mouse anti neun, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Clone g a 5, by Merck Group (1 mentions)
Mouse anti glutamine synthetase, by Merck Group (1 mentions)
Papain, by Roche (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Rabbit anti pkcα, by Santa Cruz Biotechnology (1 mentions)
Mouse anti calbindin, by Swant (1 mentions)
Rabbit anti kir4, by Merck Group (1 mentions)
Goat anti calretinin, by Swant (1 mentions)
Mouse anti adenomatous polyposis coli apc, by Abcam (1 mentions)
Mouse anti neurofilament 200 nf200, by Merck Group (1 mentions)
Mouse anti microtubule associated protein 2 map 2, by Merck Group (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Lsm 700 axioimager, by Zeiss (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
16 protocols using mouse anti glial fibrillary acidic protein
1
Immunocytochemical Analysis of Retinal Cell Types
2
Immunofluorescence Staining of Spinal Cord
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Specific proteins were detected by using immunofluorescence staining (IFS) as reported previously [41 (link)]. Briefly, the post-fixed spinal cord was sectioned at 20-μm thickness with a cryostat. The sections were rinsed with 0.01 M phosphate-buffered saline (PBS) three times, blocked with 10 % goat serum for 30 min, and incubated with primary antibodies mixed in 0.3 % Triton X-100 overnight at 4 °C. After rinsing in PBS, the sections were incubated with secondary antibodies and examined under a fluorescence microscope. A summary of antibodies used is as follows: rabbit anti-β-tubulin III (Tju-1) (1:300, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-microtubule-associated protein 2 (Map2) (1:1000, Sigma-Aldrich), mouse anti-glial fibrillary acidic protein (GFAP) (1:300, Sigma-Aldrich), mouse anti-neurofilament 200 (NF200) (1:300, Sigma-Aldrich), rabbit anti-myelin basic protein (MBP) (1:600, Chemicon, now part of Millipore Corporation, Billerica, MA, USA), and mouse anti-adenomatous polyposis coli (APC) (1:200, Abcam, Cambridge, UK).
3
Neuroinflammation and Astrocyte Activation in Neonatal Mice
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All litters from C57BL/6J mice were randomly divided into two groups: vehicle- and polyI:C-treated. Neonatal C57BL/6J mice were administered with a daily subcutaneous injection of saline (control) or polyI:C (5 mg/kg, Sigma-Aldrich) between PD2 and PD6. Immunohistochemistry was conducted as described previously [17 (link)]. Neonatal mice were deeply anesthetized with diethyl ether 24 h after the final polyI:C treatment and perfused transcardially with saline, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). The brains were removed and cryoprotected. 20-μm-thick coronal brain sections were cut on a cryostat and mounted on slides. The sections were denatured in a microwave oven in 0.01 M citrate buffer (pH 6.0). After blocking with 5% donkey and 5% goat serum/PBS, mouse anti-glial fibrillary acidic protein (GFAP, a marker for astrocytes, 1:1,000, Sigma-Aldrich) and rat anti-Fstl1 (1:100, R&D systems) were added to the sections. After washing in PBS, goat anti-mouse Alexa Fluor (AF) 568 and anti-rat AF488 antibodies (1:1,000, Invitrogen) were added to the sections. The samples were observed using a confocal-laser scanning microscope (LSM 700 Axio Imager; Zeiss).
4
Immunofluorescence Visualization of Microbead-Adherent Cells
5
Immunochemistry Staining and Quantification
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For immunochemistry, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in phosphate-buffered saline (PBS) and blocked with 10% normal goat serum (NGS) in 0.3% Triton X-100. The cells were incubated with rabbit anti-σ-1R (Invitrogen, Carlsbad, USA; 1:250), rabbit anti-p-Src (Cell Signaling, Danvers, MA, USA; 1:500), or mouse anti-glial fibrillary acidic protein (GFAP) (Sigma-Aldrich, St. Louis, MO, USA; 1:800) antibodies overnight at 4°C. Following washing three times with PBS, the cells were then incubated with AlexaFluor 488-conjugated anti-rabbit IgG or AlexaFluor 594 goat anti-mouse IgG (Invitrogen, Carlsbad, USA; 1:250) for 1 h to detect σ-1R, p-Src, and GFAP. After a final washing with PBS, the slides were mounted with ProLong Gold Anti-fade reagent to visualize the nuclei onto glass slides (Invitrogen, Carlsbad, USA). Immunofluorescence images were captured using confocal microscopy (Olympus FV100, Olympus, Tokyo, Japan). Average intensities of GFAP were calculated using Image J by sampling a 28 × 28 pixel area, 36 images taken from six consecutive sections. Values were reported as average intensity above background ± SD.
6
Immunohistochemical Analysis of Subcortical Nuclei
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Mice were fixed by transcardial perfusion with 10% buffered neutral formalin (Sigma) and 40 μm coronal cryosections spanning the subcortical striatal nuclei (including globus pallidus and caudate putamen) were collected after cryoprotection in 30% sucrose. Tissue treatment by antigen retrieval followed by immunodetection of antigens has been described elsewhere (von Jonquieres et al., 2014 (link)). Sections were treated with a combination of primary antibodies including rabbit-anti GFP or mouse anti-GFP (Roche, Switzerland) with either mouse anti-NeuN (Millipore, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP; Sigma-Aldrich, MO, USA), or rabbit anti-aspartoacylase (ASPA; Mersmann et al., 2011 (link)). ASPA is a marker of mature oligodendrocytes. In this population, ASPA positive cells have been reported to overlap 100 and 93% with the widely used oligodendrocyte soma markers glutathione S-transferase π isoform and CC1, respectively (Kawai et al., 2009 (link)). Following incubation with the appropriate Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA), sections were mounted on slides and coverslipped with Mowiol (Calbiochem, Darmstadt, Germany). Fluorescence was visualized using a Zeiss Z1 AxioExaminer NLO710 confocal microscope (Carl Zeiss MicroImaging, Germany).
7
Biochemical and Immunological Reagents for Research
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T4 DNA ligase, NdeI, and EcoRI were obtained from New England Biolabs (Ipswich, MA, USA). Alexa Fluor 488- and 594-conjugated donkey secondary antibodies were obtained from Invitrogen (Carlsbad, CA, USA). Streptozotocin (STZ), D-glucose, and some reagents for the western blot analysis such as mouse anti-glial fibrillary acidic protein (GFAP), β-actin, and α-tubulin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Primary antibodies such as GLP-1, Tau, NF-κBp65, insulin receptor (IR)-β, and calbindin were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Rabbit anti-parvalbumin, GLP-1R, hippocalcin, nuclear p84, and mitochondrial VDAC1 were obtained from Abcam (Cambridge, MA, USA). Isopropyl-β-thiogalactopyranoside (IPTG), LB broth, p-Tau, and 4′,6-diamidino-2-phenylindol (DAPI) were obtained from the Thermo Fisher Scientific (Waltham, MA, USA). Total dynamin related protein1 (Drp1) and optic atrophy1 (OPA) were obtained from BD Bioscience (Franklin Lakes, NJ, USA).
8
Immunostaining of Frozen Brain Sections
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Frozen sections collected on slides were air-dried at room temperature for 10 min and washed with PBS for 10 min, then blocked with Tris-buffered saline containing 10% donkey serum and 0.3% Triton X-100 for 1 h at room temperature. Primary antibodies in the same blocking solution were applied overnight at 4°C. Mouse anti-activated caspase-3 antibody (1:1000; Sigma) was used to detect cell apoptosis; mouse anti-adenomatous polyposis coli (CC1, 1:100; Calbiochem, La Jolla, CA, USA), mouse anti-glial fibrillary acidic protein (GFAP, 1:200; Sigma), and mouse anti-neurofilament-H (SMI-31, 1:1000; Chemicon, Billerica, MA, USA) antibodies were used to detect OLs, astrocytes and axons, respectively. The slides were washed three times in PBS and incubated with fluorescein isothiocyanate- or rhodamine-conjugated goat anti-mouse IgG (all at 1:200; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 37°C. Slides were washed three times with PBS and mounted with Gel/Mount containing Hoechst 33342 to counterstain nuclei. Images were acquired with a BX60 fluorescence microscope or Zeiss 510 laser confocal microscope (Oberkochen, Germany). Control samples were prepared by omitting the primary antibody.
9
Immunohistochemical Analysis of Neuroinflammation
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After the tests of abnormal behavior, mice were anesthetized and transcardially perfused with ice-cold PBS. Their brains were then removed and processed for optical microscopy or confocal fluorescence microscopy as previously reported [6 ]. For optical microscopy, a rabbit polyclonal antibody against ionized calcium-binding adaptor molecule 1 (IBA1; Wako, Osaka, Japan) was used as the primary antibody. The secondary antibody was EnVision-plus system HRP-labeled polymer (anti-rabbit; Dako, Glostrup, Denmark). Immunoreactivity was developed and visualized by use of DAB substrate (SK-4100; Vector Laboratories, Burlingame, CA, USA) and quantified by using ImageJ software (NIH, Bethesda, MD, USA). For confocal fluorescence microscopy, the primary antibodies used were goat anti-COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-glial fibrillary acidic protein (GFAP; Sigma-Aldrich, St. Louis, MO, USA); and the secondary antibodies were Alexa Fluor 488-labeled donkey anti-goat IgG (H + L) (Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568-labeled goat anti-mouse IgG (H + L), respectively. The mounting medium used was VECTASHIELD (Vector Laboratories, Burlingame, CA, USA). Images of the hippocampus were captured with a confocal fluorescence microscopy system (LSM510; Zeiss, Oberkochen, Germany).
10
Immunofluorescence Staining of Neural Markers
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The following primary antibodies were used: rat anti-BrdU 1:200 (Insite Biotechnology, UK) , mouse anti-rat nestin 1:200 (BD Biosciences, UK) , mouse anti-Glial Fibrillary Acidic Protein (GFAP) 1:200 (Sigma-Aldrich, UK ) , rabbit anti-GFAP 1:500 (DAKO, UK) , mouse anti-Neuron-specific class III betatubulin (TuJ1) (1:500 Sigma-Aldrich, UK) , rabbit anti-TuJ1 1:500 (Cambridge Bioscience Ltd, UK) , rabbit anti-GluR2 1:200 (Merck Millipore, UK) a nd rabbit anti-Ki-67 1:500 (Novocastra, UK) . Primary antibodies were probed using Cyanin2/Alexa 488, Cyanin3/Alexa 594, or Cyanin5/Alexa 647-conjugated anti-rat 1:500, anti-mouse 1:500 and/or anti-rabbit 1:200 secondary antibodies (Jackson ImmunoResearch, USA) . Cells were then counterstained with DAPI (5 µg/mL, Sigma-Aldrich, UK). For BrdU immunostaining, DNA was first denatured by incubating the cells in 2 M HCl at 37°C for 30 min. Omission of primary antibodies revealed no staining.
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