Primary muscle cultures (pp6) were isolated as described (Qu et al., 1998 (link)) from the indicated mouse lines. FoxO3-depleted (FoxO-KO) pp6 cells were prepared from foxO3+/− mice as previously described (Dentice et al., 2010 (link)). C2C12 cells were obtained from ATCC. In some experiments, endogenous T3 and T4 were removed from the FBS by charcoal absorption (Larsen, 1972 (link)). Transient transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Single myofibers were prepared from the extensor digitorumlongus and gastrocnemius muscles of 6- to 12-week-old mice as previously described (Rosenblatt et al., 1995 (link)). Pax7Hi-Lo isolation by FACS has been described elsewhere (Rocheteau et al., 2012 (link)). Anti-MyoD (sc-304), myogenin (sc-12732), tubulin (sc-8035), and anti-FoxO3 antibodies were purchased from Santa Cruz Biotechnology. Polyclonal anti-MHC antibody (MF-20a) and anti-Pax7 antibody were from Developmental Studies Hybridoma Bank. Anti-D3 antibody is described elsewhere (Huang et al., 2003 (link)). Anti-Desmin antibody was from MP Biomedicals (#10519). Anti-total Akt was from Upstate, and anti-pAkt and anti-PARP were from Cell Signaling Technology. TUNEL assay was performed using the ApopTag kit from Millipore. Caspase-3/Caspase-7 activity was measured by using the CaspaseGlo kit from Promega.
Anti pax7 antibody
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
The Anti-Pax7 antibody is a laboratory reagent produced by the Developmental Studies Hybridoma Bank. It is a monoclonal antibody that specifically binds to the Pax7 protein, which is a transcription factor involved in the regulation of muscle development and stem cell differentiation. The antibody can be used in various research applications, such as immunohistochemistry, Western blotting, and flow cytometry, to detect and analyze the expression of Pax7 in biological samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Caspaseglo kit, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Apoptag kit, by Merck Group (1 mentions)
C2c12 cell, by American Type Culture Collection (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Affinipure fab fragment, by Jackson ImmunoResearch (1 mentions)
Cy3 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Anti myod antibody, by Santa Cruz Biotechnology (1 mentions)
Dapi mounting medium, by Abcam (1 mentions)
Bx63 microscope, by Olympus (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti laminin antibody, by Merck Group (1 mentions)
Tcs sp5, by Leica (1 mentions)
Alexa 568, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
5 protocols using anti pax7 antibody
1
Isolation and Characterization of Primary Muscle Cells
2
Pax7 and MyoD Immunostaining Protocol
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For Pax7 immunostaining, fresh sections were fixed in 4% paraformaldehyde for 20 min, permeabilized with methanol (-20°C) for 6 min, and blocked first with a solution containing 4% bovine serum albumen (BSA; Jackson) in PBS and then with goat anti-mouse AffiniPure Fab fragment (1:100; Jackson) after antigen retrieval with 100 mmol/L sodium citrate. The sections were then incubated overnight at 4°C with an anti-Pax7 antibody (1:20; Developmental Studies Hybridoma Bank). After washing with PBS, Pax7 signals were visualized by incubating with biotin-conjugated goat anti-mouse IgG1 (1:1000; Jackson) and Cy3-conjugated streptavidin (Jackson; 1:2500). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen). For MyoD immunostaining, the fresh sections were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100/PBS (PBST) for 10 min, and blocked by incubating with 5% BSA/6% goat serum/0.2% PBST at room temperature for 2 h. Immunostaining with anti-MyoD antibody (1:50; Santa Cruz) was performed by overnight incubation at 4°C. After washing, immunoreactive proteins were visualized by incubating with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. Nuclei were stained with DAPI (Roche). The Pax7+ and MyoD+ cells in the sections (n = 10) were counted.
3
Immunostaining Myofibres for Pax7
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Myofibres were isolated as described above, fixed with 4% paraformaldehyde and immunostained with anti-Pax7 antibody (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) as previously described [12 (link)].
4
Muscle Satellite Cell Quantification
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For Pax7 immunostaining, soleus muscle sections were first labelled with anti‐Pax7 antibody (dilution at 3 μg/mL, Developmental Studies Hybridoma Bank) for 1 h, followed with AlexaFluor 555 goat anti‐mouse (1:1000, Invitrogen). After washing, slides were incubated with anti‐laminin antibody (1:100, Sigma Aldrich) and detected with an AlexaFluor 488 goat anti‐rabbit (1:1000, Invitrogen). Then, soleus muscles were counterstained with a DAPI mounting medium (Abcam). Five to ten fields were acquired with a 20× magnification using an Olympus BX63 microscope. At least 500 fibres were used to record the PAX7+/DAPI+ satellite cells and the data were normalized by the number of laminin positive fibres.
5
Immunostaining of Zebrafish Satellite Cells
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Embryos injected with mylfpa:Cas9-T2A-GFP or mylfpa:Cas9-T2A-GFP;U6:8.2 were sacrificed with MS-222 overdose and fixed in 4% paraformaldehyde (Electron Microscopy Sciences, USA) in phosphate-buffered saline (1× PBS) overnight at 4 °C, rinsed in 0.1% PBS-T (0.1% Triton X-100 in 1× PBS), permeabilized 1 h with 0.5% PBS-T and blocked 1 h with blocking buffer (2% normal goat serum, 2 mg/mL BSA, 0.1% Tween20 in PBS). Embryos were incubated overnight with an anti-Pax7 antibody [51 (link)] (1:50, monoclonal, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) diluted in blocking buffer. Embryos were then incubated with anti-mouse Alexa 568 (1:800, Invitrogen, Eugene, OR, USA) and DAPI (1:1000, Invitrogen, Eugene, OR, USA), diluted in blocking buffer. Embryos were rinsed as above and stored in 50% glycerol in 1× PBS. Microscopy slides were prepared using 50% glycerol in 1× PBS. Images were acquired with a laser point scanning confocal microscope (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany). Positive satellite cells in the central myotome and the vertical and horizontal myosepta where quantified, while xanthophores, pigment cells in the surface of somites, with higher intensity staining and bean-shaped nuclei were excluded [52 (link)].
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