Immunostaining of frozen sections was done as described previously18 (link),17 (link). Briefly, retinal explants were fixed with 4% paraformaldehyde and treated with 15% and 30% sucrose in this order, and embedded in the optimal cutting temperature compound (Sakura Fineteck). Primarily antibodies used were monoclonal antibodies against active Caspase 3 (Promega), Ki67 (BD Bioscience), HuC/D (Molecular Probes), Chx10 (Exalha Biologicals), NR2E3 (photoreceptor-specific nuclear receptor, PNR) (ppmx), glutamine synthetase (GS) (Millipore), PKC (Merck Millipore), Rhodopsin (Rho4D2, kindly donated by Dr R. S. Molday, The University of British Columbia), and a polyclonal antibody against GFP (Clontech). Nuclei were counterstained with 4′,6-diamidino-2-phnylindole, dihydrochloride (DAPI). Sections were then treated with Alexa-488-, Alexa-594-, or Alexa-680-conjugated appropriate secondary antibodies. Photos were taken under observation using Zeiss Axio Image M1 and Axio Image M2.
Glutamine synthetase
Manufactured by Merck Group
Sourced in United States
Glutamine synthetase is an enzyme that catalyzes the conversion of glutamate and ammonia into glutamine. It plays a crucial role in the regulation of nitrogen metabolism within cells.
Automatically generated - may contain errors
Lab products found in correlation
Huc d, by Thermo Fisher Scientific (1 mentions)
Active caspase 3, by Promega (1 mentions)
Axio image m1, by Zeiss (1 mentions)
Axio image m2, by Zeiss (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Mounting solution, by Vector Laboratories (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
4 protocols using glutamine synthetase
1
Immunostaining of Retinal Explants
2
Isolation and Characterization of Muller Cells
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Fresh donor eyes were obtained from Saving Sight Eye Bank (Kansas City, MO, USA), provided by Weiming Mao, Department of Ophthalmology, Indiana University. Müller cells were isolated as described previously.27 (link) Retinas were minced and incubated in 0.25% trypsin (Thermo Fisher Scientific) at 37°C for 1 hour. The tissue pieces were gently triturated by pipetting and washed in high glucose DMEM/Ham's F-12 medium (1:1 ratio, 2.25 g/L glucose) containing 20% fetal bovine serum and 1% antibiotic-antimycotic. After attachment in a high glucose medium, cells were fed with low glucose DMEM/Ham's F-12 medium (1:1 ration, 0.5 g/L glucose) supplemented with 20% FBS and 1% antibiotic-antimycotic. The Müller cells were characterized using antibodies to vimentin (Cat. #C9080, 1:200; Sigma-Aldrich Corp.), glial fibrillary acidic protein (Cat. #12389S, 1:200; Cell Signaling Technology), and glutamine synthetase (Cat. #MAB302, 1:200; Millipore Corp.). Cells used in this study were of passages 3 to 6.
3
Immunohistochemical Analysis of Retinal Cells
4
Immunofluorescence Staining of MIO-M1 Cells
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MIO-M1 cells seeded in 6-well culture plates were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min. The PFA was removed, and the plates were washed with PBS. Fixed cells were treated with blocking buffer containing 2% BSA in PBS for 1 h on ice. Plates were incubated for 24 h in primary antibodies in a humidified chamber. The following antibodies and concentrations were used: Glutamine Synthetase (1:500, Millipore, Cat. No. MABN1182, RRID: AB_2110656), GFAP (1:500, Millipore, Cat. No. G6171, RRID:AB_1840893), MAP4K4 (1:500; Cell Signaling Tec., Cat. No. 3485, RRID: AB_2140972), MAP4K6 (1:500; Novus, Cat. No. NBP1-22989, RRID: AB_1726507) and MAP4K7 (1:500; Genetex, Cat. No. GTX13141, RRID: AB_383061), YAP (1:100, Santa Cruz, Cat. No. 101199, RRID: AB_1131430), ki67 (1:500, Abcam, Cat. No. ab16667, RRID: AB_302459), HuC/D (1:500, Abcam, Cat. No. ab184267, RRID: AB_2864321). After washing with PBS, anti-mouse (488, 1:300, Thermo Fisher Scientific, Cat. No. A21022, RRID: AB_141607) or anti-rabbit (488, 1:300, Thermo Fisher Scientific, Cat. No. A11008, RRID: AB_143165 or 594, 1:300, Thermo Fisher Scientific, Cat. No. A11012, RRID: AB_141359) secondary antibodies were applied, and signals were visualized using a Zeiss LSM 880+Airyscan.
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