At the end of each experiment, the hearts were collected and fixed overnight at 4°C in 2% paraformaldehyde. They were then rinsed in PBS and equilibrated in 30% sucrose before embedding in Tissue-Tek OCT compound (Sakura Finetek Europe B.V.) and cryo-sectioned at a thickness of 16 µm. The immunohistochemistry procedures were performed as previously described [23 (link)]. The following primary antibodies were used: rabbit anti-MCM5 at 1 : 5000 (kindly provided by Soojin Ryu, Heidelberg), mouse anti-p-Histone 3 at 1 : 200 (Millipore, Clone 3H10), rabbit anti-Mef2 at 1 : 100 (Santa Cruz Biotech SC-313), rabbit anti-DsRed (Clonetech, 632496) at 1 : 200 and rat anti-BrdU at 1 : 100 (Abcam, ab6326). To visualize the cardiac muscle, sections were incubated for 30 min with Phalloidin-Atto 647N (Sigma-Aldrich) at a dilution of 1 : 500. For the BrdU immunostaining, the slides were incubated in 2 M HCl in PBS with 0.3% Triton-X for 45 min before the immunohistochemistry procedure. The Alexa-Fluor-conjugated secondary antibodies (Jackson Immunoresearch) were used at 1 : 500, and DAPI was used at 1 : 2000. Haematoxylin and eosin (H & E) staining was performed as previously described [15 (link)].
Rabbit anti mef2
Manufactured by Santa Cruz Biotechnology
Rabbit anti-Mef2 is a primary antibody that recognizes the myocyte enhancer factor 2 (Mef2) protein. Mef2 is a transcription factor involved in the regulation of muscle-specific gene expression. The Rabbit anti-Mef2 antibody can be used to detect and study the Mef2 protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti brdu, by Abcam (2 mentions)
Rabbit anti dsred, by Takara Bio (1 mentions)
Phalloidin atto 647n, by Merck Group (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Alexa fluor conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Tbs tissue freezing medium, by Thermo Fisher Scientific (1 mentions)
Anti pcna, by Thermo Fisher Scientific (1 mentions)
Mouse anti pcna, by Merck Group (1 mentions)
Biotinylated wfa, by Vector Laboratories (1 mentions)
Chabc, by Merck Group (1 mentions)
Mouse anti gad67, by Merck Group (1 mentions)
Anti pv, by Swant (1 mentions)
Pv cre b6 129p2 pvalbtm1 cre arbr j, by Jackson ImmunoResearch (1 mentions)
D ap5, by Bio-Techne (1 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Diazepam, by Roche (1 mentions)
3 protocols using rabbit anti mef2
1
Cardiac Tissue Preparation and Immunostaining
2
Cardiac Muscle Proliferation Quantification
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Hearts were extracted, fixed in 4% paraformaldehyde, equilibrated in 30% sucrose, then embedded in TBS tissue freezing medium (Fisher Scientific, Hampton, NH #TFM-C) and sectioned to 10 μm. Primary antibodies used were rabbit anti-Mef2 (Santa Cruz Biotechnology, Santa Cruz, CA #SC-313; 1:75) and mouse anti-Pcna (Sigma-Aldrich, Nadick, MA #P8825; 1:400). Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG (H + L) for anti-Mef2, and Alexa Fluor 594 goat anti-mouse IgG (H + L) for anti-Pcna (Invitrogen - Thermo Fisher Scientific, Waltham, MA #A11034, #A11020). Images were captured at 20× using an Olympus BX53 microscope and Retiga 2000DC camera. CM proliferation indices were calculated as a percentage of Mef2(+)Pcna(+) cells relative to the total number of Mef2(+) cells in a defined area adjacent to the injury.
3
Transgenic Mouse Visualization and Manipulation
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Mice and Reagents PV-Cre: B6;129P2-Pvalbtm1(cre)Arbr/J was from Jackson Laboratories. PV neurons were visualized in PV-Cre x Rosa-CAG-STOP-tdTomato mice (Silvia Arber, Basel). Antibodies, goat and monoclonal mouse anti-PV (Swant, Bellinzona) 1:5,000 (goat) and 1:500 (mouse); mouse anti-GAD-67 (Chemicon) 1:500; rabbit anti-Mef2 (Santa Cruz Biotechnology, sc-313) 1:1,000; rat anti-BrdU (abcam) 1:500; mouse anti-NeuN (Millipore) 1:200; mouse anti-Bassoon (Molecular Probes) 1:200; mouse anti-Gephyrin (Molecular Probes) 1:500; biotinylated WFA (Vector laboratories), 1:500; and aBungarotoxin, Alexa 488 conjugated (Molecular Probes, Invitrogen) 1:500. Local treatments were carried out on consecutive days, through implanted cannulas (33G, Bilaney Consultants), or intraperitoneally (i.p.) (Diazepam, Roche, 3 mg/kilogram [kg] in saline; D-AP5, Tocris Bioscience, 10 mg/kg in saline). In vivo topic injections (pharmacologically selective actuator module [PSAM] virus; ChABC, 0.08 units, Sigma; Ac-Tyr 1 ,D-Phe 2 -growth-hormone releasing factor 1-29, 300 nm, Tocris) were into dorsal (coordinates, À1.58 from bregma, 1.65 mediolateral, and 1.5 dorsoventral) or ventral hippocampus (coordinates, À2.7 from Bergma, 3.25 mediolateral, and 2.25 dorsoventral). Injected solutions were less than 200 nanoliter (nl) per day.
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