The following primary antibodies were used: glutamine synthetase (1:250; BD Biosciences), rhodopsin (1:500; Millipore), cone arrestin (1:500; Millipore), PKCα (1:250; BD Biosciences), MPP4 AK4 (1:200; homemade34 (link)), CRB1 AK2 (pH 1.5) (1:200; homemade8 (link)), CRB2 (1:2008 (link)), p120-catenin (1:100; BD Biosciences), GFAP (1:200; Dako), CD11b (1:100; BD Biosciences); PLVAP (1:200; BD Pharmingen), and VE-cadherin (1:100; BD Biosciences).
Glutamine synthetase
Manufactured by BD
Sourced in United States
Glutamine synthetase is an enzyme that catalyzes the reaction between glutamate and ammonia to form glutamine. It plays a crucial role in nitrogen metabolism and is essential for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Phospho β catenin, by Cell Signaling Technology (2 mentions)
Phospho yap, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Rhodopsin, by Merck Group (1 mentions)
Cone arrestin, by Merck Group (1 mentions)
P120 catenin, by BD (1 mentions)
Plvap, by BD (1 mentions)
Ve cadherin, by BD (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Cd11b, by BD (1 mentions)
Isolectin b4, by Vector Laboratories (1 mentions)
Control igg, by BioLegend (1 mentions)
Gad65, by Abcam (1 mentions)
Streptavidin alexa 488, by Thermo Fisher Scientific (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cy3 conjugated goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Dapi containing mounting medium, by Southern Biotech (1 mentions)
Eclipse c1 microscope, by Nikon (1 mentions)
7 protocols using glutamine synthetase
1
Immunohistochemical Analysis of Retinal Cell Markers
2
Protein Extraction and Analysis from Mouse Liver
3
Protein Extraction and Analysis from Mouse Liver
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Mice were sacrificed at different time points as described. Liver tissues were removed, snap frozen in liquid nitrogen, and stored at −80 °C until use. For protein extraction, tissues were homogenized in a cold lysis buffer (50 mM Tris-HCl, pH 7.4; 1% NP-40; 0.5% sodium deoxycholate; 150 mM NaCl; 1 mM EDTA) containing protease and phosphatase inhibitor cocktails (Roche). After homogenization, the tissue lysates were allowed to solubilize for 1 h at 4 °C with rotation, and then were centrifuged at 19,700g for 30 min at 4 °C. The supernatants were used for immunoblot analyses. Nuclear extract was performed by using nuclear and cytoplasmic extraction kit (Pierce). Western blots were performed following procedure according to different antibodies. 25 to 50 ug of protein was loaded for each lane and transferred to PVDF membrane. Protein expression was analyzed by immunobloting with β-catenin (Cell Signaling), phospho-β-catenin (Cell Signaling), β-actin (Cell Signaling), H3 (Cell Signaling), p21 (BD pharmingen), p53 (kindly provided by Dr. Loning Fu), phospho-Yap (Cell Signaling), Yap (Cell Signaling), Glutamine Synthetase (BD pharmingen). All dilutions are 1:1000, except that H3 antibody dilution is 1:5000 and Glutamine Synthetase antibody dilution is 1:3000. Full gel scans are shown in Supplementary Fig. 11 .
4
Detecting Cell Signaling Pathways in CNV Model
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Antibodies used for this study include rabbit antibodies against VEGFR2 (Cell Signaling Technology, Danvers, MA, USA) and GAD65 (Abcam, Cambridge, MA, USA), goat antibody against Centaurin A (Santa Cruz Biotechnology, Dallas, TX, USA), mouse antibodies against rhodopsin (Millipore, Burlington, MA, USA) and glutamine synthetase (BD Bioscience, Franklin Lakes, NJ, USA). To neutralize VEGF in the mouse laser-induced CNV model, a mouse-specific antibody against VEGF (512807; BioLegend, San Diego, CA, USA) and the IgG control (400515; BioLegend) was purchased from the same company. Aflibercept was obtained from the eye clinic adjacent to the Department of Ophthalmology at UIC. Other reagents used were isolectin B4 (Vector Laboratories, Burlingame, CA, USA), Alexa 594 anti-rabbit antibody, Alexa 488 anti-mouse antibody, Alexa 488-streptavidin (Invitrogen, Carlsbad, CA, USA), and HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA).
5
Immunofluorescence Analysis of Liver Cryosections
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Tissue cryosections (8 µm) were washed with PBS and fixed in 100% methanol for 10 min at room temperature. After three washes with PBS, the tissue sections were blocked in blocking buffer (3% BSA, 0.3% Triton™ X-100 in PBS) for 30 min at room temperature and then incubated with primary antibody overnight at 4 °C. The next day, the tissue sections were washed with PBS and incubated with fluorescence-conjugated secondary antibodies for 1 h at room temperature. DAPI-containing mounting medium (Southern Biotech #0100–20) was used to visualize the nuclei and preserve slides. The antibodies used were as follows: CYP8B1 (Affinity, DF4762; 1:200 dilution), Glutamine synthetase (BD Biosciences, #610517; 1:200 dilution), Cy3-conjugated Goat anti-rabbit IgG (Servicebio, GB21303; 1:200 dilution), and 488-conjugated Goat anti-mouse IgG (Servicebio, GB25301; 1:200 dilution). Images were acquired using a Nikon Eclipse C1 microscope and analysed using ImageJ software.
6
Immunohistochemical Profiling of Intestinal Cells
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IHC was performed on formalin-fixed intestinal sections. Standard IHC techniques were used throughout this study. Primary antibodies used for immunohistochemistry were as follows: BrdU (1:200, BD Biosciences #347580), SOX9 (1:500, Chemicon #AB5535), β-catenin (1:50, BD Biosciences #610154), BCL9 (1:500, Abnova #H00000607-MO1), Glutamine Synthetase (1:200 BD Biosciences #610518), γ-H2AX (1:50, Cell Signalling Technologies #9718) and CD44 (1:50 BD Biosciences #550538). For each antibody, staining was performed on at least three mice of each genotype, representative images are shown for each staining. For nuclear β-catenin staining Tris-EDTA based antigen retrieval was used.
7
Quantifying Ductular Reaction in Liver Biopsies
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Immunostains for cytokeratin 7 (CK7) (Dako, Carpentaria, CA, USA) and glutamine synthetase (BD, San Jose, CA, USA ) were performed on two consecutive sections of each biopsy case utilizing an automated stainer (Bond automated IHC stainer). The degree of ductular reaction within each glutamine synthetase-positive centrilobular zone of every biopsy was recorded as either: 0: no CK7+ cells, 1: isolated single CK7+ cells, 2: CK7+ cells in strings, or 3: CK7+ ductular structures (Figure 1). In addition every portal tract in the CK7 stained slides was graded as either: 0: no ductular reaction, 1: mild ductular reaction, or 2: florid ductular reaction.
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